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Molecular Characterization of Photoperiodic Flowering in Strawberry (Fragaria sp.)


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1 MOLECULAR CHARACTERIZATION OF PHOTOPERIODIC FLOWERING IN STRAWBERRY ( Fragaria SP.) By PHILIP JACOB STEWART A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA May 2007

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2 2007 Philip J. Stewart

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3 To my parents, who always believed in me, and taught me to love knowledge and the natural world. To the memory of my grandfather, Jackson Lee Stewart, Jr., who introduced me to agriculture, and who will forever be my image of the American farmer. To my children, Zea and Adina, who have been a constant inspiration and joy to me, and reminders of the things in life that are truly important. And, most of all, to my wife, Cynthia, who has been unwavering in her faith in my abilities, in her willingness to sacrifice for my academic and professional pursuits, and, most importantly, in her love.

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4 ACKNOWLEDGMENTS I would like to take this opportunity to thank at least a few of the many people who have have assisted me along the path to this degree. First, I would like to offer my love and gratitude to my parents, Lee and Faith Stewart, and my in-laws, Barton and Teri Cowan, all of whom have provided continuous support, both emotional and financial, throughout the course of my graduate studies. None of this would have been possible without them. Secondly, I would like to thank those that who have provided invaluable assistance in my academic and professional development, in particular my graduate advisors, Kevin Folta and Craig Chandler. Their help was critical to the work described here. I am also grateful for the assistance and suggestions offered by the other members of my committee: Paul Lyrene, Natalia Peres, and Jos Chaparro, all of whom helped to shape this study, and Daniel Sargent, of East Malling Research, U.K., for the use of his diploid mapping population. Much thanks also go to Dawn Bies and Maureen Clancy, for technical assistance, and also to my fellow students in the Folta lab: Denise Tombolato, Stefanie Maruhnich, Thelma Madzima, and Wendy Gonzalez. I’ve enjoyed working with all of you and wish you much success as you finish your studies and move into professional life. And, finally, the greatest debt of gratitude is owed my beautiful wife, Cynthia, who has not only been tolerant but encouraging as I have dragged her from place to place across the country in pursuit of this dream. Thank you so much—I hope it was worth it.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...........................................................................................................4 LIST OF TABLES................................................................................................................. .....8 LIST OF FIGURES................................................................................................................ ....9 ABSTRACT....................................................................................................................... ......11 CHAPTER 1INTRODUCTION .............................................................................................................12 2LITERATURE REVIEW...................................................................................................14 The genus Fragaria ...........................................................................................................14 Polyploidy in Strawberries.................................................................................................15 Flowering Habits in Strawberry.........................................................................................16 Short-Day Stra wberr ies ...............................................................................................16 Remontant Strawberries..............................................................................................18 Factors Affecting Expression Of Photoperiodic Fl owerin g.................................................23 Temperature Effects On Flower ing.............................................................................23 Vernalization Requi rements ........................................................................................24 Juvenility and Plan t Age E ffects ..................................................................................25 Growth Pattern Differences Between Plan ts Of Differing Fl owering Ha bits .......................26 Inheritance Of Flowering Habit In Stra wberry ................................................................... 27 Molecular Markers For Flowering Habit............................................................................32 Molecular Control Of Flow ering In Arab idopsis ................................................................33 Components Upstream of CO ......................................................................................34 Components Downstream of CO .................................................................................35 The CONSTANS-LIKE Gene Family .........................................................................36 Components of the Flowering Pathwa ys are Conserve d Among Sp ecies .....................38 Using Model Systems To Unders tand Developmen t In Ros aceae ................................40 3CHARACTERIZATION OF CONSTANS -LIKE GENES IN Fragaria ..............................44 Introductio n................................................................................................................... ....44 Results........................................................................................................................ .......46 Cloning And Identification Of Four CONSTANS-LIKE Genes Of Fragaria ...............46 FrCO / FrCOL Gene Structure......................................................................................48 Southern blot analysis of FrCO Copy Numb er............................................................ 50 Mapping Of Fragaria CO And COL Genes In A Diploid Mapping Popul ation ...........50Comparison Of Strawberry Genotype s Under Varying Photoperi ods ...........................51 Allele-Specific Expression Patterns Differ Betwen Short-Day And Day-Neutral Genotypes...............................................................................................................52

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6 Expression Of Other Genes Unde r Different P hotoperi ods .......................................... 53 Some FraCOL2 Transcripts Contain An Unspliced Intron...........................................54 Discussion..................................................................................................................... ....54 The Co Gene Family In Fragaria (Rosaceae) ..............................................................54 Evolutionary Relationships Among FrCO Sequences..................................................58 FrCO Expressi on ........................................................................................................ 61 Expression of Other COL Gene s.................................................................................65 Materials and Methods ....................................................................................................... 68 Plant M aterial .............................................................................................................68 Plant Growth Conditi ons .............................................................................................69 Cloning of FraCO .......................................................................................................70 Identification of COL Gene s in Gene bank...................................................................70 Gene Structure Characterizatio n..................................................................................70 Southern Blot Analysis...............................................................................................71 Genetic Linkage Mapping COL genes in Fragaria ......................................................71 Extraction of Nu cleic Acid s........................................................................................72 Reverse Transcription and RTPCR...........................................................................73 4IDENTIFICATION AND CHARACTERIZATION OF FLOWERING-RELATED GENES IN Fragaria AND OTHER ROSACEAE .............................................................93 Introductio n................................................................................................................... ....93 Results........................................................................................................................ .......94 Identification of Rosaceae Flower ing Gene Ho mologs ................................................94 FrSOC1 Expressi on.................................................................................................... 97 FrTFL1 and FrLFY Expressi on................................................................................... 97 Discussion..................................................................................................................... ....98 Rosaceae MADS-Box Genes ......................................................................................98 The FT/TFL1 Gene F amily in Ros aceae ....................................................................101 Other Flowering Genes.............................................................................................105 FrSOC1 FrLFY and FrTFL1 Expressi on................................................................. 105Methods and Materials ..................................................................................................... 109 Identification of Rosaceae Fl owering Ge nes..............................................................109 Phylogenetic Tree Constructio n................................................................................ 109 RNA Extraction and RT -PCR Exper iments ...............................................................109 5 Fragaria ALLELES OF POLYGALACTURONASE INHIBITOR PROTEIN GENES...118 Introductio n................................................................................................................... ..118 Results........................................................................................................................ .....121 Identification of Fragaria PGIP Allele s....................................................................121 Amino Acid Similar ities Among Alleles ................................................................... 123 Discussion..................................................................................................................... ..123 Methods and Materials ..................................................................................................... 126 Plant and Gen etic Mater ial ........................................................................................126 Cloning and Sequencing of Fragaria PGIP A lleles ...................................................127 Sequence Analysis....................................................................................................127

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7 LIST OF REFERENCES........................................................................................................132 BIOGRAPHICAL SKETCH...................................................................................................151

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8 LIST OF TABLES Table page 3-1 Comparison of amino acid identity (%) of the predicted protein encoded by FraCO to those of Group Ia CONSTANS-LIKE genes in various species. ....................................75 3-2 RT-PCR primers for strawberry CONSTANS-like genes and controls, sequence source, TM, and approximate size in cDNA (as calculated from sequence). ....................76 3-3.GenBank accession numbers for Fragaria COL genes and other Rosaceae orthologs....77 3-4 Mean inflorescence and runner production of F. ananassa and F. vesca genotypes under LD (16 h) and SD (8 h) photoperiods, at 18/16C day/night temperat ure. .............86 3-5.Sources of plants used in this chapter ............................................................................ 90 4-1 Identified Fragaria genes related to flowering and photoperiodic response, with the closest match at the amino acid level among Arabidopsis and all annotated transcripts.................................................................................................................... 112 4-2RT-PCR primers for strawberry flowering genes and controls, sequence source, TM, and approximate size in cDNA (as calculated from sequence). ....................................114 5-1 FaPGIP alleles identified in various octoploid strawb erry cultiv ars. ............................. 129 5-2Percentage of identity between PGIP isoform s at the mRNA and amino acid lev els. ....130

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9 LIST OF FIGURES Figure page 2-1Simplified diagram showing interactions of genes and environmental factors governing flowering in Arabidopsis thaliana .................................................................43 3-1Cladogram of CO-like genes, including all Arabidopsis and full-length Rosaceae COL genes as well as all those from other species Group Ia genes demonstrated to functionally complement Arabidopsis co mutants..........................................................78 3-2Alignment of the predicted amino acid sequence for FraCO with those of AtCO and functionally confirmed dicot homologs, w ith major conserved domains no ted. ..............79 3-3Alignment of the predicted amino acid sequence for FrvCOL1 with possible apple and Arabidopsis homologs, with major conserved do mains noted ..................................80 3-4Structures of COL alleles from Fragaria species: F. ananassa ‘Strawberry Festival’ ( Fra ), F. vesca FDP815 ( Frv ), F. nubicola FDP 601 ( Frn ), and F. iinumae FRA377 ( Fri ).............................................................................................................................. 81 3-5NJ phylogram tree of intron sequence divergence among FrCO alleles. .........................82 3-6Comparisons of hypervariable section of coding region in FrCO ...................................83 3-7Southern blot of genomic DNA from F. ananassa ‘Strawberry Festival’ and F. vesca Hawaii 4, cut with six different enzymes and probed with a portion of FrvCO .....84 3-8Mapping of FrCO in a reference diploid popu lation. ..................................................... 85 3-9Northern blot of RNA from ‘Strawberry Festival’, a SD genotype, and ‘Diamante’, a DN genotype, collected every 4 h, showing expression of FraCO under short and long day cond itions. ...................................................................................................... 87 3-10RT-PCR assay of expression of FrCO transcript under short (8 h) and long (16 h) days in three genotypes: F. vesca Hawaii-4 (SD, diploid), F. ananassa ‘Camarosa’ (SD, octoploid), and F. ananassa ‘Diamante’ (DN, octoploid)....................................88 3-11Expression of 35 and C9 alleles of FraCO at dawn under SD, using RT-PCR at 33 and 40 cycles, in ‘Strawberry Fes tival’ (F) and ‘D iamante’ (D )..................................... 89 3-12RT-PCR assay of FrCOL2 expression in F. vesca Hawaii-4, showing variation in splicing efficiency at several points under short days ..................................................... 91 3-13Ubiquitin controls after equalization of RT-PCR samples using RN-Ubiq1 primer set to standardize template amount s....................................................................................92

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10 4-1NJ tree showing similarity between full-length amino acid sequences of Rosaceae and Arabidopsis MADS-Box genes .............................................................................111 4-2NJ phylogeny tree showing relationships between Arabidopsis and Rosaceae members of the FT/TFL1 gene family, base d on full predicted prot ein seque nce. ........ 113 4-3RT-PCR assay of expression of FrSOC1 under short (8 h) and long (16 h) photoperiods in ‘Camarosa’ (SD, octopl oid) and ‘Diamante’ ( DN, octopl oid). ............ 115 4-4RT-PCR assay of expression of FrLFY under short (8 h) and long (16 h) photoperiods in Hawaii-4 (SD, diploid) and ‘Camarosa’ (SD, octopl oid). .................... 116 4-5RT-PCR assay of expression of FrTFL1 under short (8 h) and long (16 h) photperiods in Hawaii -4 (SD, di ploid). ........................................................................ 117 5-1Amino acid alignment of the polymorphic portion of a number of Fragaria PGIP genes. Shaded areas indicate th e xxLxLxx beta-sh eet regions ......................................131

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11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MOLECULAR CHARACTERIZATION OF PHOTOPERIOD FLOWERING IN STRAWBERRY ( Fragaria ) By Philip J. Stewart May 2007 Chair: Craig Chandler Cochair: Kevin Folta Major: Horticultural Science The initiation of flowering is a critical developmental transistion for most plant species, affecting reproductive potential and evolutionary success. The timing of this event in strawberry ( Fragaria sp.) is conditioned by a number of factors, including photoperiod. Sensitivity to photoperiod varies among strawberry genotypes, which are generally divided into three categories: short-day, everbearing, and day-neutral. The genetic basis for these variations is not known, but existing model plant systems such Arabidopsis thaliana and rice reveal a wellconserved network of genes and proteins governing the perception of photoperiod and the regulation of floral initiation. A number of these genes were identified and characterized in strawberry and other relatives in the family Rosaceae, and their expression in strawberry under long and short photoperiods assayed. Expression profiles suggest that while many of the critical genes governing photoperiodic flowering are conserved between strawberry and model species, their expression and relationships to one another are unlike those of any previously characterized plant species.

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12 CHAPTER 1 INTRODUCTION The initiation of flowering is among the most important developmental transitions a plant makes, second perhaps only to germination. Mistimed flowering endangers the reproductive potential of plant in a number of ways. A plant that flowers too early or too late may find the seasonal conditions not conducive to seed development, or yield seeds that germinate at points in the season that make seedling survival difficult or impossible. In cross-pollinated species even a difference of a few days may be adequate to render reproduction impossible, putting a plant out of syncronization with others of its species. Any of these difficulties has the potential to render an individual an evolutionary dead end. Because of this necessity, plants have developed an extraordinary regulatory network allowing the fine tuning of the transition to flowering by a range of environmental and internal factors. In the model eudicot Arabidopsis, these factors have been shown to include the environmental inputs of photoperiod, light quality, and temperature, and internal pathways controlled by the hormone gibberellic acid and autonomous factors such as developmental age. At the molecular level, each of these inputs represents separate elements of genetic pathways that converge at a few floral integrators to regulate flowering through a common set of meristem identity genes. These integrators convert the complex and heterogeneous inputs from the various environmental and internal cues into a decision to flower, influencing a choice made in a small cluster of meristmatic cells, and driving development from a vegetative to reproductive state through the activation of genes that remodel cell fate. Key among these floral integrators is CONSTANS ( CO ), which serves to tie the perception of photoperiod with that of the spectral quality of light received.

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13 Flowering in cultivated strawberry ( Fragaria ananassa Duch.) has historically received a great deal of attention from researchers, for two primary reasons. First, strawberry is an attractive platform for flowering research because despite a narrow genetic base (Sjulin & Dale, 1987) it exhibits at least three distinct and ranging flowering habits: junebearing (short day), dayneutral (photoperiod insensitive), and everbearing (often referred to as “long day” but more accurately another distinct form of photoperiod insensitivity). These clear phenotypic delineations in such a genetically homogeneous background suggests fairly simple genetic difference between markedly contrasting flowering habits. Secondly, in an increasingly global market where fruit is readily transported not only across countries but around the world, farmers depend on a precise understanding of flowering behavior to time harvest to coincide with the best market windows in order to obtain the best price for their fruit. In an era of narrowing profit margins, this timing may be the difference between a profitable crop and a loss.

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14 CHAPTER 2 LITERATURE REVIEW The genus Fragaria All strawberry species belong to the genus Fragaria, and are members of Rosaceae, a family that contains a large number of economically significant crops, primarily fruits such as apple ( Malus domestica Borkh.), pear ( Pyrus sp.), stone fruits ( Prunus sp.), and brambles ( Rubus sp.), but also ornamentals such as rose ( Rosa sp.). Strawberries belong to the Rosoideae subfamily, which includes the genera Rubus, Rosa, Potentilla and Duchesnea Authorities vary somewhat on the exact number of strawberry species, but most recent authors list about twenty species (Hancock and Luby, 1993; Folta and Davis, 2006). Native strawberries occur in much of North America, Europe, and Asia, as well as parts of South America, and Hawaii (Darrow, 1966; Hancock and Luby, 1993). Cultivated strawberry, F. ananassa Duch., is a species of relatively recent hybrid origin, being derived from the accidental hybridization of two New World octoploid species, F. virginiana Duch. and F. chiloensis (L.) P. Mill., beginning in France some time in the late 1700s (Darrow, 1966). Although the polyploid nature of the crop and its hybrid origin have assured that the species contains some genetic diversity, the actual germplasm base used is relatively small, with only 53 founding clones (Sjulin and Dale, 1987) and 17 initial cytoplasm donors (Dale and Sjulin, 1990) contributing to the cultivated varieties. Some recent introgressions from the wild have been made, most notably from a selection of F. virginiana ssp. Duch. var. glauca (S. Wats) Staudt, but in general very little new genetic material has been added since the early years of strawberry breeding (Darrow, 1966).

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15 Polyploidy in Strawberries As has been previously mentioned, the cultivated strawberry is an octoploid (2n=8x=56). The majority of strawberry species, however, are diploids, and there exist tetraploid and hexaploid taxa as well (Folta and Davis, 2006). Considerable evidence suggests that the cultivated strawberry is an alloploid, with either three or four distinct subgenome types. The earliest model, that of Federova (1946), proposed a composition of AABBBBCC, but this was replaced with AAA A BBBB when Senanayake and Bringhurst (1967) found evidence of partial homology between the A and C genomes. Mounting evidence of diploidization (Byrne and Jelenkovic, 1976; Arulsekar, et al. 1981) prompted Bringhurst (1990) to propose a fully diploidized model: AAA A BBB B The origins of the component genomes have long been a subject of debate. As early as 1926, Ichijima suggested that one of these might be derived from F. vesca based on cytogenetic observations of F. virginiana (8x) x F. vesca L. (2x) hybrids, in which the formation of seven bivalents was noted. Further data, including cytology (Senanayake and Bringhurst, 1967) and sequence analysis of the chloroplast trnL-trnF region (Potter et al., 2000), the nuclear sequences ITS (Potter et al., 2000) and polygalacturonase inhibitor protein (PGIP) (Chapter 5 of this work), have strengthened this view, and the A genome is now widely believed to be derived from F. vesca (Folta and Davis, 2006). The origins of the other genomes have been less clear. The study of ITS and trnL-trnF sequences implicated F. nubicola as a possible A-type genome donor, in addition to F. vesca (Potter et al., 2000). Recent work by DiMeglio and Davis (unpublished but cited in Folta and Davis, 2006) examining the alcohol dehydrogenase locus implicates two other species, F. mandshurica Staudt and F. iinumae Makino. Potter et al. (2000) and Harrison et al. (1997) found

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16 F. iinumae to be the most distantly related of all other Fragaria species examined, and as such may represent the ancestor of one or both of B and B’ genomes. Unlike most diploid species, which possess at least a degree of compatibility with other diploid Fragaria (Dowrick and Williams 1959), F. iinumae is not compatible with F. vesca (Bors, 2000; Sargent et al., 2004b), and displays a karyotype distinct from the other diploid species (Iwatsubo and Naruhashi, 1989, 1991), which may account for the failure of B genome chromosomes to pair with those of the A genomes. F. mandshurica on the other hand, is a newly described diploid species (Staudt, 2003) not included in previous studies. It is however thought to be related to the tetraploid species F. orientalis Losinsk., to which it bears a strong resemblance. Potter et al. (2000) had placed F. orientalis in the A clade, and thus F. mandshurica may represent A’ genome in the octoploid species. Flowering Habits in Strawberry Although many authors (Fuller, 1897; Fletcher, 1917) had suggested the presence of environmental influences on flowering in strawberry, Sudds (1928) was perhaps the first to note the specific effects of photoperiod and temperature on flower bud induction in strawberry. Soon after, Darrow and Waldo (1929, 1930, 1933) conducted an exhaustive series of experiments, classifying over a hundred octoploid strawberry genotypes as flowering under either short or long days, finding the groups to generally coincide with what were already termed “June bearers” and “ever bearers”. Short-Day Strawberries It is believed that the natural state of most strawberry species is as a short day (SD) plant (Darrow, 1966), though there is considerable variation in senstivity. It appears that the earliest cultivated octoploids all flowered under short days (Darrow, 1966), although the limits of the required photoperiod vary considerably between genotypes. In general, most SD genotypes form

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17 flower buds under photoperiods of less than 14 h, which was first suggested by Darrow (1936). Three major factors seem to dictate response to photoperiod: genotype, temperature, and chilling. Genotype effects on photoperiodic flowering in SD strawberries Darrow concluded that photoperiods of 9.5 to 13.5 h induced the greatest number of flowers in SD cultivars, with an optimum around 12 h (1936). While still generally true, further studies and perhaps continued progress in breeding have widened this range somewhat. On one end of the range, some authors (Izhar, 1997; Faedi et al., 2002) have delineated a class of strawberries they refer to as “infra short-day.” These plants flower under longer day lengths (typically 13.5-14 h photoperiod) and with little chilling requirement, but seem to represent merely an extension of the existing spectrum of sensitivities already known in SD plants, rather than a distinct flowering habit. In other genotypes, photoperiods of considerably less than 12 h are required for optimal flowering. In a study by Heide (1977) ‘Abundance’ demonstrated peak flowering under a 10 h photoperiod, the shortest tested in the study. A number of studies have shown robust flowering under 8 h photoperiods in some cultivars (Sonsteby, 1997; Leshem and Koller, 1964; Barger et al. 1997; Moore and Hough, 1962). Many early researchers compared short and long day lengths in terms of natural light versus natural light plus an artificial extension of day length, making it difficult to assign specific photoperiods. It is interesting to note that while SD photoperiods are needed for optimal floral induction, they may actually delay floral development. Several studies of SD cultivars have shown that continuing SD photoperiods may actually slow the development of previously initiated buds compared to longer photoperiods (Moore and Hough, 1962; Durner and Poling, 1987). Consequently, Salisbury and Ross (1992) consider strawberries to be SD for purposes of flower induction, but LD plants for flower development. A similar relationship for temperature has also been noted (see below).

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18 Remontant Strawberries Alpine strawberries The first major exception to this flowering pattern noted was the alpine strawberry, F. vesca var. semperflorens Duch. Found at several places in the European Alps, these represent a mutation of the common diploid European wood strawberry, and were among the earliest types to be cultivated. Though first mentioned as early as 1553, they gained prominence in the mid 1700’s when they were introduced into France and England, possibly from Turin, Italy (Duchesne, 1766). In 1811, a runnerless form was obtained by Labaute (Darrow, 1966). Now only occasionally cultivated, these varieties are of only minor commercial importance (Darrow, 1966), although in the early nineteenth century a white-fruited variety was extensively cultivated in Quebec (Fletcher, 1917). Fairly little work has been done to characterize the floral physiology of these varieties, and the fact that many workers have studied unnamed genotypes that are either seed-propagated or even collected directly from the wild (Chabot, 1978) makes it difficult to consistently track genotype-specific differences. Sironval and El Tannir-Lomba (1960) found that their selection of F. vesca var. semperflorens required days longer than 12 h to form flower buds, and that SD treatments inhibited flowering in plants that had previously reached the flowering stage under LD conditions, though this appears to be the exception, not the rule (Darrow, 1966). As in other strawberries, temperature has a significant effect, with moderately low temperatures favoring both reproductive development and biomass production (Chabot, 1978). “Everbearers” Relatively quickly after the introduction of octoploid cultivars, however, so-called “everbearing” (EB) strawberries were identified (Darrow, 1966). Such genotypes initiate flowering most heavily under the long days of summer, and often on unrooted or newly rooted

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19 runners, resulting in what is in most locations primarily a fall harvest, rather than an early summer one, or in some cases two distinct crops. These genotypes appear to trace to a few distinct sources within cultivated types. suggesting spontaneous mutation of a single gene. Development of such types took place independently in both North American and Europe, but very little crossover has ever occurred between these two sources of the trait. The first North American EB cultivar described was possibly ‘Oregon Everbearing,’ obtained in 1882, although Fletcher (1917) describes an everbearing selection of what was likely F. virginiana found in Ohio in 1852. The first successful everbearer, however, was likely ‘Pan-American,’ an apparent sport of the June bearer ‘Bismarck’ found in 1898 (Fletcher, 1917). ‘Pan-American,’ in turn, became the source of the trait for a number of other successful everbearing cultivars, including ‘Progressive’ and ‘Rockhill’ (Darrow, 1966). This last selection is the source of the trait in the proprietary everbearing cultivars developed by Driscoll Strawberry Associates (Darrow, 1966). Other sources of the trait in North America include ‘Gem,’ a sport of ‘Champion’ introduced in 1933 that proved quite popular for several decades. That the trait is the result of instability at a single locus is supported by documented reversion to short day flowering in the case of ‘June Rockhill’, a short day mutant of the everbearer ‘Rockhill’ (Darrow, 1966). European octoploid everbearers, called “ remontant ” by the French, may have had their origin somewhat earlier. Unlike the American sources of the trait, which can largely be traced to mutations identified in cultivated plants, the source of the European everbearers is unclear. Although claimed at the time to be hybrids of large-fruited octoploids with the everbearing Alpine diploids (Fletcher, 1917), it seems doubtful that this was truly the case. First, such hybrids of F. vesca with the octoploids are difficult to make (Yarnell, 1931; Mangelsdorf and

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20 East, 1927) and even more difficult to restore to adequate fertility, and secondly the inheritance of the trait in the octoploids seems to be different than in Alpine strawberries (Brown and Wareing, 1965; Clark, 1937). More likely, accidental cross-pollination resulted in seedlings of pure F. ananassa pedigree, among which was selected the original everbearer. Some, such as ‘Louis Gauthier,’ a double-cropping French cultivar, may have been simply SD genotypes with unusually permissive photoperiod or chilling requirements (Fletcher, 1917). Richardson (1914) suggests that the first true European octoploid everbearer was ‘Gloede’s Seedling,’ introduced in 1866, and this may be the source for most or all European everbearers that followed (Ahmadi et al., 1990). ‘Mabille’ and ‘l’Inepuisable,’ considered by some (Fletcher, 1917) to be pure Alpine strawberry, followed this, but none were commercially successful. A French priest, Abbe Thivolet, was among the first to breed for this trait, and after more than a decade of breeding both octoploids and diploids (Fletcher, 1917) introduced a series of everbearers, culminating in ‘St. Joseph’, which Darrow (1966) refers to as the “first true large-fruited everbearer.” Although a considerable improvement over the preceding everbearers, ‘St. Joseph’ and Thivolet’s later ‘St. Antoine de Padoue’ were not especially productive, but spurred the development of later improved types. While EB plants derived from this source remain in production today, this source has contributed very little to American breeding. Although they have been called “long day” cultivars, these varieties lack the characteristic behaviors associated with true long day plants, in the sense that they do not appear to be regulated by the length of the dark period, and do not react identically to a short day with a brief night break of light as they do to long photoperiods (Dennis et al., 1970; Durner et al. 1984), nor is flowering inhibited by SD (Durner et al., 1984). Rather, these genotypes seem more dependent on the total amount of light received. Dennis et al. (1970) found no difference in flower

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21 formation in the everbearing cultivar Geneva grown under 12 h photoperiods versus those grown under a 10 h photoperiod with a 2 h night break, a treatment which often inhibits flowering in SD plants (Downs and Piringer, 1955; Piringer and Scott, 1964). Marked increases, however, were seen with 18 and 24 h photoperiods. Additionally, they saw a significant effect of light intensity when increased from 1200 to 2400 f-c on the 18 and 24 h photoperiod treatments (increases were also seen on the 12 h and 10 + 2 h treatments, but these were not statistically significant). This study and the later study by Durner et al. (1984) both noted similar levels of flowering for both 12 h photoperiods and 10 + 2 h night break treatments, suggesting that amount of light, not duration of either darkness or light, is the critical factor. “Day-neutrals” The third distinct flowering habit is day-neutral (DN). Observers of wild American octoploids had noted that many plants of F. virginiana subsp. glauca (then called F. ovalis ) flowered in the summer and fall when most wild and commercial short day cultivars had stopped (Darrow, 1966). Researchers with the USDA evaluated a large number of F. virginiana subsp. glauca selections in the 1930s and 1940s, and a many were included in breeding efforts at the time. A number of everbearing varieties resulted from these efforts, including two, ‘Arapahoe’ and ‘Ogallala,’ (Hildreth and Powers, 1941) that are still in existence today. Whether the trait expressed in these cultivars was the older EB trait or the DN trait derived from glauca is unknown, as sources of both are present in the pedigrees, the strong tendency of most remontant seedlings of ‘Arapahoe’ and ‘Ogallala’ to fruit in their first year is distinct from those other EB parents (Ourecky and Slate, 1967), and more typical of DN genotypes (Ahmadi et al., 1991). Regardless, the introduction of the DN trait that has had the largest impact came in the 1970’s, when the University of California breeding program, under Royce Bringhurst and Victor Voth, utilized a single selection of F. virginiana subsp. glauca from the Wasatch Mountains in Utah to

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22 introduce this habit into commercial strawberries (Bringhurst and Voth, 1984). Strawberries derived from this source constituted a majority of the cultivars planted in California in 1999 (Hancock, 1999). These were distinct from the everbearing habit in that the plants were truly insensitive to the length of the photoperiod received, and fruit at nearly the same rate over a broad range of photoperiods (Durner et al., 1984; Ahmadi et al., 1991). Day-neutral cultivars offered improved heat tolerance and longer harvest seasons compared with the earlier everbearers, but runner poorly and are difficult to propagate (Durner et al., 1984). Although day-neutrals appear to differ from older everbearing types, many researchers have failed to distinguish between them, and in some cases use the terms interchangeably, resulting in confusion. For the purposes of this work, “day-neutral” (DN) will refer to the trait derived from F. virginiana ssp. glauca, while “everbearing” will refer to that derived from ‘Pan-American’, ‘Gloede’s Seedling,’ or other older sources, and the generic “remontant” will be used to refer to cases where both are implied or the author has not been clear in distinguishing. Other flowering habits Arguably a fourth flowering habit, poorly characterized at this time, is the amphiphotoperiodic behavior exemplified by the F. chiloensis selection CHI24-1 (Yanagi et al. 2006). This genotype behaves as a standard SD plant under most conditions, with long photoperiods inhibiting flowering, but initiates flowers under continuous lighting as well. Although many will cease flowering altogether (Thompson and Guttridge, 1960), a few cultivated SD strawberry cultivars may fall into this category, as both ‘Sparkle’ (Collins and Barker, 1964) and ‘Sweet Charlie’ (unpublished observations) have been observed to flower under such conditions as well, but thus far little research has been done on this phenomenon.

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23 There has been much interest in recent years in finding novel sources of remontancy, and workers have identified a number of wild accessions that seem to display varying degrees of photoperiod-insensitivity or continual flowering, mostly among F. virginiana (Sakin et al., 1997; Hancock et al., 2001, 2002; Sere and Hancock, 2005a, b). These new sources have not yet been adequately characterized to determine whether they fall neatly into the DN or EB categories, or whether they represent truly novel mechanisms. Inheritance studies by Sere and Hancock (2005a) suggest that there may be multiple genetic mechanisms at work. Factors Affecting Expression Of Photoperiodic Flowering Temperature Effects On Flowering A relationship between temperature and flower initiation was established by early workers, and was highlighted in the large studies of flowering by Darrow and Waldo (1929, 1930, 1933). Research has also shown that lower temperatures may permit flowering under longer daylengths even in normally SD cultivars (Darrow and Waldo, 1934; Darrow, 1936). Hartmann (1947b) concluded that this was the reason that cultivars grown in the coastal district of California near Watsonville, where the mean temperature is 17C, were able to flower and fruit through the long days of summer, while the same cultivars planted inland near Sacramento, where the mean temperature was 23C, behaved as strict short day plants and produced only a spring crop. In greenhouse studies he found that plants of four cultivars important to the region all flowered under long days at 17C, but none flowered at 23C. Three of the four same genotypes flowered freely under both temperatures under short day conditions, while a fourth, ‘Fairfax,’ failed to flower at all under the higher temperature, even under 10 h days. Even everbearing cultivars under long days are inhibited by high temperatures, as are day-neutral types, though in general the critical temperature is higher for DN cultivars (Durner et al., 1984; Manakasem and Goodwin, 2001). Heide (1977) found two of four Scandinavian SD cultivars still flowered under

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24 photoperiods of 14, 16, and even 24 h when grown at 12C or 18C, though flowering was substantially decreased under the longer photoperiods. The effects were highly dependent on genotype, however, particularly in the middle of the temperature range, a consistent finding of such studies. In some cases cultivars that are considered to be short day in some locations may behave as remontant at other locations ‘Climax’, a British selection, behaved like a SD plant when grown in the United States, but as a two-crop variety in England where temperatures are coller and summer days are longer (Downs & Piringer, 1955). Much like SD photoperiods, which are necessary to develop flower buds, but inhibit the development of flower trusses, low temperatures allow for flower induction under long photoperiods, but may not be optimal for fruit production, because lower temperatures slow development of the flower trusses (Darrow, 1966). Le Miere et al., (1996) found optimum temperatures for truss development to be about 19C in ‘Elsanta’. Temperatures that fluctuate diurnally seem to be more effective in promoting flower development than continuous temperatures, even at the same average temperature (Hartmann, 1947a). Such thermoperiodic rhythms have been shown to have similar effects in other species (Seneca, 1974; Alvarenga and Vlio, 1989) and may allow for more robust entrainment of the circadian clock (Salom and McClung, 2005). Vernalization Requirements Temperature also plays a second role in the expression of flowering, by conditioning the plant for flowering before the season begins. A certain amount of chilling, generally defined as time between 0C and 7C, may be required to break bud dormancy and proceed with the normal cycle of development, though the extent of chilling required is highly cultivar-dependent (Piringer and Scott, 1964; Voth and Bringhurst, 1970; Durner and Poling, 1986; Darnell and Hancock, 1996). Increased chilling has been shown to correlate with increases in leaf area and

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25 number, petiole length, and runner initiation (Guttridge 1969; Bringhurst et al., 1960; Piringer and Scott, 1964; Braun and Kender, 1985; Lieten, 1997). Chilling has been shown to promote both vegetative and reproductive development (Darnell and Hancock, 1996). Durner and Poling (1987) found chilling to enhance vegetative growth and reduce flower induction while promoting floral differentiation. Non-chilled plants produce fruit of smaller size (Harmann and Poling, 1997) and lower quality (Bringhurst et al., 1960) than do the identical genotypes with adequate chilling. Because of the related decrease in flower induction, however, chilling beyond the required amount is of no advantage and can even markedly reduce the number of flowers. Piringer and Scott (1964), for example, saw substantial decreases in flower number on ‘Marshall’ with increasing chilling. Juvenility and Plant Age Effects Juvenility may also play a role in the expression of photoperiod-sensitivity. In general, young plants devote more resources to vegetative than reproductive growth, and may not flower at all during this portion of their life cycle. This allows a plant to attain sufficient size to support fruit and seed development before flowering (Thomas and Vince-Prue, 1984) Ourecky and Slate (1967), working with the EB trait, noted that when populations were retained for a year and scored for a second season, more plants were determined to be everbearing (a finding supported by unpublished worked cited in Scott & Lawrence (1975)), casting a degree of doubt on earlier work that utilized only first year data. However, Ahmadi et al. (1990) found that DN progeny generally flowered within four months of germination. While not an issue for plants in cultivation, this effect may have serious impacts on the ability of breeders to make selections based on flowering habit. Ito and Saito (1962) found that older plants responded more robustly to

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26 both photoperiod and temperature. This may have been in part a function of plant size, although Hartmann (1947a) found that a single intact leaf was enough to perceive inductive photoperiods. Growth Pattern Differences Between Plants Of Differing Flowering Habits In addition to the differences in flower production, strawberries of differing flowering habits also differ in terms of plant architecture and growth patterns. The production of runners varies considerably among SD, DN, and EB genotypes. Runner initiation is affected by photoperiod and temperature as well. In SD cultivars, there appears to be a balance between runners and inflorescences ranging from almost no runners and many flowers under optimal SD conditions, through an intermediate area where flowers and runners coexist, and then solely runnering under the longest days. SD plants will runner in response to short days with a night break, and they tend to shift from reproductive to vegetative growth under high temperatures (Piringer and Scott, 1964; Durner et al., 1984). Interestingly, DN cultivars, while flowering in a photoperiod insensitive manner, seem to retain a sensitivity to photoperiod in respect to runnering, with the number of runners increasing under long days, night break, or high temperature conditions. This is in contrast to EB types, which do not show photoperiodic or temperature changes in runner initiation (Durner et al., 1984). In many, though not all cases (Durner et al., 1984) EB genotypes runner very little, which may be a barrier to efficient propagation. Nicoll and Galletta (1987) also noted differences in plant architecture between DN, EB, and SD plants. Under long days, the main axis of SD and weakly DN plants remains vegetative and most axillary buds develop as runners, while under short days, the main axis terminates in an inflorescence and an upper axillary bud develops as a branch crown. In EB plants, under all photoperiods, most or all axillary buds that develop do so as branch crowns, rather than runners, and it is the termination of these crowns with inflorescences that results in most flowering,

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27 though the main axis will occasionally terminate in a flower and continue growth from a side shoot as in SD plants. In DN plants, by contrast, the growth is characterized by very low rates of branch crown formation, with upward growth quickly terminated by a terminal inflorescence and continued from an upper bud, with reduced development of leaves, runners, and branch crowns. Interestingly, these growth habits closely parallel those seen in F. vesca by Brown and Wareing (1965), but in that case were found to associate closely with the runnering locus, or a gene close by it, rather than the seasonality locus, described below. The runnerless gene from ‘Baron Solemacher’ conveyed a growth pattern like that observed in DN plants, while runnerless plants descended from ‘Bush White’ had a many-crowned habit like that of EB octoploids, and the wild type F. vesca was similar to the SD octoploids. Inheritance Of Flowering Habit In Strawberry Inheritance of photoperiod insensitivity has been described in a number of species. Most commonly, it is a single gene trait, conferred by either a dominant allele, as in rice ( Oryza sativa L,) (Chandrartna, 1953), sweet pea ( Lathyrus odoratus L.) (Ross and Murfet, 1985), jute ( Corchorus sp.) (Joshua and Thakare, 1986), and tetraploid Sea Island cotton ( Gossypium barbadense ) (Lewis and Richmond, 1960), or a recessive allele, as in diploid upland cotton ( Gossypium hirsutum L.) (Lewis and Richmond, 1957), cucumber ( Cucumis sativus L.) (Dellavecchia and Peterson, 1984), or okra ( Abelmoschus esculentus (L.) Moench) (Wyatt, 1985). In a few instances, the inheritance has been shown to be a more complicated mechanism, such as the two dominant alleles at separate loci responsible in some hexaploid wheat ( Tritcum aestivum L.) (Maystrenko and Aliev, 1986) or the three genes believed to be involved in sesame ( Sesamum indicum L.) (Kotecha et al. 1975). Brown and Wareing (1965) demonstrated that seasonality in F. vesca is conveyed by a single gene, which they designated S (referred to as SFL by some later authors (Albani et al.,

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28 2004)), with the perpetual flowering habit displayed by the homozygous recessive. They further demonstrated that the trait was independently inherited from the non-runnering habit, which proved to be another recessive trait at a different locus. Federova (1937) may have identified seasonality in F. vesca as a recessive trait earlier, but it is unclear with which species he was working. Ahmadi et al. (1990) expanded on this by observing that the California F. vesca which differs significantly from the European F. vesca morphologically, appears to have two additional genes conveying photoperiod sensitivity, yielding a 1:63 ratio in the F2 of a cross between Alpine F. vesca and local California plants. Richardson (1914) conducted some of the first studies of the inheritance of the EB trait. Using ‘St. Antoine de Padoue’ and ‘Laxton’s Perpetual’, both derived from ‘St. Joseph’, as sources of the trait, he found ratios of 1:1 and 3:1, suggesting a simply inherited character. A considerably different model was that of Clark (1937), who investigated the expression of the EB trait from ‘Mastodon’, an everbearer derived from ‘Pan-American’, in breeding populations grown in New Jersey. In general, there was a strong trend towards producing approximately one third everbearers in crosses of EB with non-everbearers (presumably SD), with an average of two-thirds in crosses of EB x EB, whereas Mastodon selfed yielded 80% EB, though in a small population. Clark also cites an earlier EB x EB cross, reported by Macoun (1924), which yielded slightly less, 56.29% EB. In nearly all cases, crosses of SD x SD, in which one of the parents had one EB parent, yielded no EB progeny. There were, however, exceptions. One SD plant with an EB parent, N.J. 220, did give some EB offspring in one cross. Additionally, two everbearers, N.J. 1 and N.J. 8, produced very low numbers of everbearers in their progeny in crosses with non-everbearers (0% and 8.8%, respectively) and when selfed (0% and 11.9%, respectively). Clark concluded that the results suggested a complex,

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29 polygenic inheritance, but with evidence that the character is largely, though not wholly, conveyed by a major, dominant allele, possibly modified by other genes. Later work attempted to clarify this model, although with limited success. Powers (1954) performed a partial diallel crossing of three EB and seven SD genotypes and developed a three locus model with four dominant alleles, A’, A, B, and C conveying the everbearing trait with varying strength, ranging from A’ > A > B > C Any of these alone was theorized to be inadequate for expression of the trait, as was aaB_C_ but A’ or A plus a dominant allele at either of the other loci resulted in the everbearing character. He also theorized that as many as four recessive genes account for the presence of EB plants in the SD x SD progenies. Although mostly adequate to explain the observed data (though 5 of his 49 families do exhibit high chi-square values with this model), Powers’ model assumed that all seven of the SD parents in his study were of the same genotype, aaBbCc Unless there is a significant unknown selective value to the heterozygote at the B and C loci, it seems exceedingly unlikely that all three unrelated short day genotypes would be double heterozygotes. Two confounding factors may have been at work here. First, the everbearing parents used are primarily breeding selections, so the source of the trait cannot be readily ascertained. Since the Cheyenne USDA breeding program was utilizing F. virginiana ssp. glauca in addition to EB cultivars as parents, it is possible that Powers’ observations are the result of a mingling of DN and EB sources. Secondly, Powers used the production of flowers in July, August, or September as criteria for EB. In light of the fact that Powers’ experiments were conducted in the field at Cheyenne, Wyoming, it may be that low temperatures allowed flowering to continue even during the long days of summer. Mid-summer day length in Cheyenne is slightly less than 15 h, whereas the 1971-2000 mean monthly temperatures for Cheyenne in June, July, August, and September

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30 were 16.4, 19.8, 18.8, and 13.6C, respectively (National Climatic Data Center, 2001). Heide (1977) found that at 18C, three of four SD cultivars still initiated at least some flowers under 14 h days, and thus Powers may have, in fact, been observing everbearing behavior in noneverbearing genotypes. Later studies may have been affected similarly. Ourecky & Slate (1967) examined 46 progenies and suggested complementary dominant genes segregating in an octoploid manner. This is somewhat at odds with later work suggesting that octoploid strawberries are diploidized (Arulsekar et al. 1981), despite recent hybrid origin. More recent work by Sugimoto et al. (2005) found a 1:1 ratio of EB : SD in a cross of ‘Ever Berry’ (EB) x ‘Toyonaka’ (SD), as well as in its reciprocal, whereas ‘Ever Berry’ selfed gave 3:1 and ‘Toyonaka’ selfed gave all SD progeny. This is in line with earlier work using ‘Ever Berry’ as a parent (Monma et al., 1990; Igrashi et al., 1994), and implies a simple monogenic dominant inheritance. Although most authors have considered all sources of the EB trait to be genetically identical, it may be worth noting that these Japanese studies, as well as Richardson’s early studies (1914), the only studies to show clear monogenic ratios, have used cultivars that probably carry the everbearing trait derived from the European source (or sources). The studies which have obtained multigene or confusing inheritance ratios have all used American sources of the trait: Clark (1937), used ‘Mastodon’, derived from the ‘Pan-American’ source; Ourecky and Slate (1967) used a wide range of cultivars derived from either ‘Pan-American’ or ‘Streamliner’ (a seedling of unknown pedigree); and Powers (1954), as previously mentioned, may have been using a mix of EB and DN parents. Thus the traits, while similar, may have slightly different genetic mechanisms, or the American sources possess contain modifying genes not found in the European cultivars.

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31 Characterization of the inheritance of the day-neutrality trait has been clouded by the fact that studies have indiscriminately included “everbearers” from both the ‘Pan-American’ source and true day-neutrals from the F. virginiana ssp. glauca source. At first, day-neutrality was thought to be the result of a single dominant gene. Ahmadi et al. (1990) found this in an inspection of nearly 30,000 progeny of crosses between day-neutral and short day plants over the course of five years. These results firmly agree with a single-gene hypothesis, showing significant deviation from the model in only one family during one year. Importantly, unlike early work on everbearers, there were no seedlings that expressed the trait among progenies from SD x SD. Although the study clearly deals primarily with the glauca source, it is not clear which remontant cultivars were used and whether all derive from glauca However, their results differ significantly from later studies that did include EB from the ‘Pan-American’ source, which suggests that all four of the DN cultivars used are derived from glauca Sere and Hancock (2005a) also studied the inheritance of day-neutrality, looking at both cultivated DN and EB cultivars, and apparent novel sources of remontancy found in wild F. virginiana Segregation ratios varied widely, apparently suggesting inheritance more complex than a single gene model. A similar study by Shaw and Famula (2005) using 45 cultivated genotypes thought to derive their photoperiod insensitivity from the F. virginiana ssp. glauca source, though not identified in the paper, found strong evidence for the presence of a major dominant locus for day-neutrality, with putative homozygotes flowering more robustly under LD conditions than heterozygotes. However, it was noted that even homozygous DN plants were not wholly true-breeding for the trait, suggesting either the effects of other minor loci or deviations from diploid inheritance.

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32 The system used for scoring progenies may have had a significant impact on the results of such studies. A primary difference between Richardson’s (1914) studies and many of those that followed is that Richardson considered everbearers to be individuals that continued flowering through October, rather than stopping at the end of the summer. Ahmadi et al. (1990) considered this a more accurate identification of remontant genotypes, but theirs appears to be the only other study to have done this, as all others reviewed here continued ratings only into August or midSeptember. Sere and Hancock (2003) evaluated five methods of scoring populations for remontancy, including flowering within 100 days of germination, flowering before a specific date in the field, flowering under both long and short days in greenhouse or field, and flowering on newly formed runners in the field. Scoring by flowering within 100 days of germination was not a good predictor of remontant flowering in the field, but greenhouse observations, if conducted over the course of an entire season, were well correlated to field performance. Molecular Markers For Flowering Habit The ability to screen seedlings for flowering habit at a very early age would be of great benefit to breeders, saving the time and resources required to plant out and evaluate seedlings lacking the desired habit. A number of attempts have been made to develop such a system, but none has seen wide application in breeding. Albani et al. (2004) used inter-simple sequence repeat (ISSR) markers to identify three DNA products associated with seasonal flowering in 1,049 plants of a F. vesca ssp. vesca ( SFL/SFL ) x F. vesca ssp. semperflorens ( sfl/sfl ) BC1 population, and successfully converted these to sequence characterized amplified region (SCAR) markers. Two of these markers were linked to the seasonal flowering locus at 1.7 and 3.0 cM, while a third, SCAR2, was mapped to the same location as SFL This represents the most tightly associated marker for flowering habit yet developed in Fragaria but while this work constitutes an important research tool, the impact

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33 of these markers on practical breeding has been limited because of the differences between this trait and the DN and EB traits of the cultivated octoploid, as well as the lack of commercial importance of F. vesca Kaczmarska and Hortynski (2002) identified a single randomly amplified polymorphic DNA (RAPD) marker through bulk segregant analysis of a small F1 population, segregating 1:1 for remontancy; however, no attempt was recorded to discern how closely linked this marker was with the trait or how reliable it was across other progenies. The previously cited study by Sugimoto et al. (2005) attempted to identify RAPD markers linked to the EB trait. Five were identified; however the linkages, ranging from 11.8 to 24.3 cM, were rather weak. These weak linkages, coupled with the difficulties sometimes encountered when trying to reproduce RAPD markers (Paran and Michelmore, 1993), may limit the practical use of these markers. Molecular Control Of Flowering In Arabidopsis Although the effects of genetic variation on flowering, even allelic variation at a single locus, could be clearly seen in many species, the underlying system often proved complex, making it difficult to elucidate the roles of individual genes clearly. Among the first steps towards such an understanding was made through the development of mutant lines of Arabidopsis thaliana, (a long day (LD) annual species), displaying aberrant flowering patterns. Reinholz (1945) noted differences in flowering among irradiated Arabidopsis seedlings, and Rdei (1962) followed up on this work by treating imbibed Arabidopsis seeds with X-rays. He selected four mutants that exhibited altered timing of flowering, designating them constans ( co ), luminidependens ( ld ), and gigantea ( gi-1 gi-2 ). All of these lines flowered significantly later than the wild type control under long days, although the co plants flowered slightly faster under short photoperiods.

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34 Subsequent work has shown co to be an important element of a complex network of regulatory pathways governing flowering which are linked specifically to the perception of photoperiod. There are three distinct parts to photoperiod perception in Arabidopsis: photoreceptors that perceive light, an internal oscillator that approximates a 24 h cycle, and the output path to the meristem identity genes involved in flower initiation (Simpson, 2003). CO itself lays at the junction between the inputs of light quality and photoperiod on one side, and on the other side a series of genes, such as FT and SOC1 directly upstream of the meristem identity genes. A simplified diagram of this network is shown as Figure 2-1. Components Upstream of CO The perception of photoperiod begins with photoreceptors. Two groups, the phytochromes ( PhyA, B, D, and E ) and the cryptochromes ( Cry1 and 2 ), react to red/far-red and blue light, respectively, to entrain the complex feedback loop of the plant’s central oscillator (Millar, 2003). The central oscillator, in turn, produces a number of rhythmic outputs, including CO expression (Surez-Lpez et al., 2001). This rhythm regulates the base expression level of CO ; however, CO protein abundance is greatly influenced by a number of factors. Key among these are the further effects of photoreceptors on transcript and protein stability. PhyB has been shown to promote the degradation of CO protein under red light early in the day, while in the evening phyA counteracts the effects of phyB and stabilizes CO protein when activated under far-red light, as do the cryptochromes under blue (Valverde et al. 2004). Another apparent blue light receptor, FKF1, has been shown to be required to produce the peak in CO transcript level at the end of the day required to trigger flowering in Arabidopsis (Imaizumi, et al. 2003). A member of the same family of proteins as CO, CONSTANS-LIKE9, also appears to repress expression of CO though the mechanism is currently unknown (Cheng and Wang, 2005), as does the very similar CONSTANS-LIKE10 (Cheng and Wang, 2006).

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35 Components Downstream of CO CONSTANS acts directly on two major downstream flowering components, Flowering Locus T ( FT ) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( SOC1) (Samach, 2000). Both ultimately act to trigger a suite of meristem identity genes, (in fact many of the same genes), leading to flowering, and either may be adequate to trigger flowering (Samach, 2000), though this is disputed (Yoo et al., 2005). In addition to photoperiodic control through CO, SOC1 also responds to signals from the other pathways regulating flowering in Arabidopsis, the autonomous, temperature, and gibberellin pathways (Samach, 2000; Moon et al., 2003), and acts as an integrator of these independent inputs with the common set of meristem genes downstream. While FT and SOC1 act largely in parallel in inducing flowering (Moon et al., 2005), FT has been shown able to activate SOC1 to an extent itself (Yoo et al., 2005). Of the two, FT appears more strongly induced by the photoperiod pathway, while SOC1 is under greater control of the autonomous pathway and regulated primarily by the gene Flowering Locus C (FLC) rather than by CO (Moon et al., 2005). In Arabidopsis, another gene, TWIN SISTER OF FT ( TSF ), which is very similar to FT, has also been shown to perform similar functions (Yamaguchi et al., 2005) although there is evidence of temperature effects on both genes’ activities, suggesting that TSF is more active at low temperatures whereas FT is more active at higher temperatures (Blzquez et al., 2003) Primary among downstream genes is LEAFY (LFY) which is activated by SOC1 and triggers a number of genes controlling floral development, including AP1 (Nilsson et al., 1998). FT however, bypasses LFY and activates AP1 directly (Nilsson et al., 1998), as well as other meristem identity genes such as SEPALLATA3 (SEP3) and FRUITFULL (FUL) (TeperBamnolker and Samach, 2005).

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36 Acting as a counterbalance to FT is TERMINAL FLOWER1 ( TFL1 ). TFL1 acts to suppress the reproductive transition in the meristem, repressing expression of AP1 TFL1 is a member of the same family of genes as FT and TSF and Hanzawa et al. (2005) demonstrated that a change of a single amino acid residue in TFL1 is adequate to shift its function to that of a promoter like FT, though the resulting protein is a weaker promoter of flowering than FT TFL1 expression is elevated in COoverexpressor lines, and may in fact mirror CO ’s promotion of FT expression to an extent (Simon et al., 1996). Loss of function tfl1 mutants are early flowering and the inflorescences, unlike the indeterminate growth pattern of wild type Arabidopsis, end in a terminal flower which is often characterized by abnormal floral organs (Bradley, 1997). The CONSTANS-LIKE Gene Family CO is the best understood of a large family of genes, all of which share some basic characteristics. Specifically, all encode proteins which possess a highly conserved region of 129 amino acids called the CCT (“CO COL and TOC”, after the three classes of genes that possess it) domain near the C-terminal end, as well as two zinc-finger type domains side by side at the other end, though the exact amino acid composition of these domains varies. There are also four small internal domains present in the canonical CO protein that are retained to some extent in various other members of the COL family. All are transcription factors, though their exact mode of action is still unknown. Griffiths et al., (2003) characterized this family in Arabidopsis, rice, and barley, and found that three general groups were discernable in all three species, along with a fourth in the two monocot species. They were able to further subdivide this first group into three subgroups. The first of these, Group Ia, contains CO itself and a few closely related genes, specifically COL1 and COL2 in the case of Arabidopsis. These Arabidopsis genes have been shown to be unable to complement co mutants, and thus are not functionally interchangeable with CO but do

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37 have effects on circadian rhymicity of leaf movements and expression of cab2 transcript (Ledger et al., 2001). Many species, including Populus deltoides and P. trichocarpa (Yuceer, 2002) and Brassica nigra (Lagercrantz et al., 2002), seem to possess multiple members of this subgroup, but it is not known whether these other members are functionally equivalent to CO This study represents the first published documentation of Group Ia COL genes within the Rosaceae. Group Ib COL genes are known only in monocots (Griffiths et al., 2003), but Group Ic genes, represented by COL3/4/5 in Arabidopsis, are represented in nearly all species investigated (Jeong et al., 1999; Griffiths et al., 2003; Hecht et al., 2005). They differ from the Group Ia genes primarily in the middle region of the gene, where they lack the characteristic M2 and M3 domains (Griffiths et al., 2003). The role of these genes is poorly understood, but at least one, COL3 has been shown to have effects on photomorphogenesis, flowering, and inflorescence development (Datta et al. 2006). Zobell et al. (2005) found members of this family to be the only COL genes present in the primitive plant Physcomitrella patens suggesting that they may be the most ancient representatives of the family. Two Group Ic genes from apple, possibly paralogs resulting from alloploidy in the species’ distant past, represent the only COL proteins previously characterized in the Rosaceae (Jeong et al., 1999). Unfortunately, the authors did not take into account the diurnal expression pattern of many COL genes when documenting the expression of the genes, so it is difficult to derive much meaningful expression information from their work. Another Rosaceae Group Ic gene, designated PrpCo, was mapped in a peach-almond mapping population (Silva et al., 2005), but did not correspond to any known quantitative trait loci (QTLs) for flowering time in almond and was not further characterized. Group Ib, Id, Ie, and IV COL genes appear to occur only monocots, but Group II and III genes are present in Arabidopsis and other dicots. The functions of these groups of genes are still

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38 imperfectly known. Very little information on Group II genes appears to be available. A group III gene, AtCOL9, has been shown to play a role in flowering, apparently through the suppression of CO and, as a result, FT (Cheng & Wang, 2005). Early work presented by the same authors also showed a related gene, AtCOL10 seems to have similar effects(Cheng & Wang, 2006), and both appear to be expressed in a circadian fashion. A Group III gene from perennial ryegrass, LpCOL1 has also been shown to be under oscillator control and is implicated in vernalization (Ciannamea et al., 2006). Components of the Flowering Pathways are Conserved Among Species In some respects, Arabidopsis, a long-day annual, might seem a poor model for flowering in strawberry, a normally short-day perennial in an entirely different taxonomic family. However, while there are no doubt critical differences in the physiology of these two species, mounting evidence suggests that the pathways regulating flowering are remarkably well conserved between taxa, even those separated from Arabidopsis by considerable evolutionary distance. The components of the photoperiod and floral pathways in rice, a short day monocot species, have proven nearly identical to and interchangeable with those of Arabidopsis (Izawa et al., 2003). CO has been identified in a number of other species. Besides the model species of Arabidopsis and rice, a number of studies have suggested that homologs of CO fulfill comparable roles in other species. These include the dicots Brassica nigra L. (Robert et al., 1998), Japanese morning glory ( Pharbitis nil (L.) Choisy) (Liu et al., 2001), and potato ( Solanum tuberosum L.) (Martnez-Garca et al., 2002; Beketova et al., 2006) as well as monocots such as rice (Yano et al., 2000), common wheat (Nemoto et al., 2003), perennial ryegrass ( Lolium perenne L.) (Armstead et al., 2005; Martin et al., 2005), and barley ( Hordeum vulgare L.) (Griffiths et al., 2003). In some, although the behavior of CO itself appears to be the same,

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39 completely different photoperiod-dependent processes are being regulated. In potato, tuber development and other associated morphological changes rather than flowering (Martnez-Garca et al., 2002; Rodriguez-Falcon et al., 2006). Although modern potato cultivars have been selected for relative insensitivity to day length, some wild species such as Solanum demissum and S. tuberosum ssp. andigena are strictly dependent on short days for tuber formation (Ewing and Struik, 1992). whereas in aspen ( Populus trichocarpa ) CO regulates both flowering and the processes of bud set and growth cessation in response to shortening days in the fall (Bhlenius et al., 2006). Expressed homologs of CO are also known in species with no known photoperiodicity, such as apple (for example, GenBank accession EB141172 and others) and tomato (Drobyazina and Khavkin, 2006), though their function, if any, remains unclear. Not only is CO well conserved among species, but so are most of the other components of the system regulating flowering, both upstream and downstream. Even rice, a short-day monocot, contains homologs of nearly all the major components described in Arabidopsis (Izawa et al. 2003). Similar pictures are emerging in poplar (Bhlenius et al., 2006; Hsu et al., 2006; Mohamed, 2006), potato (Rodriguez-Falcon et al., 2006), and apple (Wada et al., 2002; Kotada and Wada, 2005; Kotada et al., 2006) and this seems likely to be the case in most plant species. Although the molecular basis for the variation in flowering habit seen in Fragaria is still unknown, such variations in other crops have been shown to related directly to alterations in this small group of critical genes. Researchers in Japan have demonstrated that two major QTL affecting heading date, Hd1 and Hd3a, correspond to orthologs of CO and FT respectively (Yano et al., 2000; Kojima et al. 2002), whereas a third heading-date QTL, Hd9 has been found to be tightly linked to the rice homolog of SOC1 (Tadege et al., 2003). Photoperiod insensitivity in barley has been shown to result from disruptions in the circadian oscillation of CO caused by

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40 mutations in Ppd-H1 a member of the pseudo-response regulator class of genes involved in circadian clock function (Turner et al., 2005) Using Model Systems To Understand Development In Rosaceae Research to date has provided a detailed understanding of the gross physiology of flowering and development in fruit crops such as strawberry, while work in models such as Arabidopsis and rice have provided most of what is known about the mechanisms at work at a molecular level. To truly capitalize on these bodies of work it is necessary to find ways of integrating information from both. This is already beginning to occur in apple. Studies have identified and characterized apple orthologs of many of the components known in Arabidopsis, including AFL1 and AFL2 (apparent equivalents of LFY ), MdAP1, MdTFL1 MdFT and over a dozen MADS-Box transcription factors involved in reproductive development (Sung et al., 1999; Yao et al., 1999; Kotoda et al, 2000, 2002; Wada et al., 2002; Kotoda and Wada, 2005). In general, such research has shown the components in apple to play roles similar to the homologous Arabidopsis genes. MdAP1 and the pair of LEAFY homologs, MdLFY-1 and MdLFY-2, have been found to promote flowering in transgenic Arabidopsis, and appear to be involved in the promotion of flowering in apple as well (Kotoda et al., 2000; Wada et al., 2002). Similarly, MdTFL1 has been shown to retard the transition to flowering in transgenic Arabidopsis (Kotoda and Wada, 2005) and suppression of MdTFL1 through expression of antisense construct of the gene has been demonstrated to shorten the vegetative juvenile phase of ‘Orin’ apple trees from more than six years to as few as eight months (Kotoda et al., 2006). Some studies, however, have shown that mechanisms in apple behave differently than would be predicted by the Arabidopsis model. For example, overexpression of AFL1 in apple, unlike overexpression of the similar LFY in Arabidopsis, does not result in precocious flowering (N. Kotoda, unpublished research cited in

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41 Kotoda et al., 2006). Regulation of LFY in Arabidopsis is primarily controlled by photoperiod (Samach, 2000) and apple, an apparently day-neutral species, may regulate flowering through a pathway not primarily dependent on LFY orthologs. Classical physiology has already offered numerous hints of what may be occurring in strawberry at the molecular level. Several researchers (Vince-Prue and Guttridge, 1973; Thompson and Guttridge, 1960) have suggested that the evidence supports the existence of a phloem-mobile inhibitor of flowering, synthesized in the leaves. This is reminiscent of the inhibitory effects of CO in rice (Yano et al., 2002), on FT which along with TFL1, has been shown to move from the leaves to the meristem in Arabidopsis (An et al., 2004). Similarly, the effects of light quality on flowering as shown by Vince-Prue and Guttridge (1973) suggest the possibility of phytochrome-mediated regulation of a hypothetical CO homolog. To truly understand what is occurring within the plant in each of these cases, however, will require the development of the needed molecular tools. The well-documented complex network of factors affecting flowering in strawberry: photoperiod, light quality, temperature, vernalization, all have corresponding mechanisms documented in the molecular models. These mechanisms may not prove identical in strawberry, and in some cases may prove radically different, yet this knowledge can act as a foundation for investigations into the interactions between genes and environment that control vital aspects of the crop’s development. To date little such work has been conducted in strawberry. Yet in many ways strawberry represents an attractive target for such studies—it has clearly delineated flowering phenotypes, a short life cycle, ease of hybridization, and convenient plant size. The octoploid genome number and alloploid nature of the cultivated species represent challenges, but the many diploid relatives

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42 have much simpler and smaller genomes. Like apple, strawberry is an important crop species, and a better understanding of the genes critical to its development might have economic impacts. Such knowledge might lead to improved cultivars and more efficient cultural practices, benefiting the farmer, consumer, and environment.

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43 Figure 2-1. Simplified diagram showing interactions of genes and environmental factors governing flowering in Arabidopsis thaliana (arrows represent promotive effects, bars represent repressive effects). LIGHTPhotoreceptors Circadian oscillatorCONSTANS (CO) SOC1 FT Meristem Identity Genes transcriptionpost-translational stability and localization Temperature FLOWERING LFY Gibberellin Autonomous TFL1

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44 CHAPTER 3 CHARACTERIZATION OF CONSTANS-LIKE GENES IN Fragaria Introduction The initiation of flowering is a critical developmental milestone in most plant species and represents a vital process for both natural evolution as well as crop production and development. The mechanisms involved in switching between vegetative and reproductive development have been shown in model species to be precisely regulated by a number of pathways, governed by both internal and environmental factors. Among these is photoperiod, which is critical to the growth and development of many crop species, including the cultivated strawberry. As in many other species, the timing of flowering in strawberry is an important factor with major impacts not only on the adaptation of cultivars to particular locales or cultural methods, but also, because of the relationship with the timing of the subsequent harvest, plays a vital role in determining the market value of the crop as well. Despite a narrow genetic base (Sjulin & Dale, 1987), commercial strawberries display a wide spectrum of sensitivities to photoperiod, ranging in phenotype from strictly short-day (SD) and “long-day” everbearing (EB) cultivars to day-neutral (DN) types that flower regardless of photoperiod. Because these different types share overwhelmingly similar genetic backgrounds and may even co-exist in the progeny from a single cross (Ahmadi et al., 1991), the differences are likely attributable to the qualitative and quantitative attributes of a small number of critical genes, thus strawberry provides a unique model system to investigate this important pathway. The ability to characterize the molecular basis for these differing phenotypes could have important implications for cultural techniques and cultivar development not only in Fragaria but also potentially in the other cultivated members of the Rosaceae, for which timing of flowering is also a critical factor.

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45 Studies in strawberry allow direct translation of molecular paradigms devised in long-day and short-day model systems to a single species with ranging photoperiodic flowering habits. Studies in Arabidopsis have shown that the gene CONSTANS ( CO) plays a critical central role in the regulatory pathway responsible for photoperiodic flowering (Simpson and Dean, 2002; Corbesier and Coupland, 2005), acting as a dynamic link between photoreceptors, the circadian oscillator, and the genes governing meristem-identity. Mutations in the CO gene cause delayed flowering in Arabidopsis, a LD plant (Putterill et al., 1995), and early flowering in rice, a SD plant (Yano et al. 2000). Its function as a regulator of flowering is well-conserved across species, having been characterized in both other eudicots (Robert et al., 1998; Liu et al., 2001) and monocots (Yano et al., 2000; Martin et al., 2004; Nemoto et al., 2003), though the downstream effect varies between species, either suppressing or promoting flowering. CO is one member of a large family of CONSTANS -like genes, numbering 17 in Arabidopsis, 16 in rice ( Oryza sativa ) and at least nine in barley ( Hordeum vulgare ) (Griffiths et al. 2003). No accounting of these genes appears to have been previously published in strawberry. The high degree of conservation in both structure and function, even between monocots and eudicots, suggests that COL genes likely have important roles in controlling flowering time across species. A similar situation is likely the case within Fragaria yet its diverse flowering habits suggest that regulation may be imparted through novel alleles of known genes, uncharacterized patterns of expression, or pathways that circumvent photoperiodic influence by acting on elements downstream from the photoperiodic regulators. This study proposes to identify, catalog and characterize probable COL genes in Fragaria describe tissue specific, temporal and photophysiological expression patterns. The results of this study provide additional

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46 information about into the mechanisms that regulate photoperiodic development. These results have implications for the evolution of the gene family in Rosaceae. Nomenclature Because of the difficulties in coordinating the numbering of family members across species that may possess different numbers of such genes, a decision was made to simply number the COL genes in the order they were found, rather than attempting to match numbering of Arabidopsis genes. In this report, FrCOL is used to refer to genes at a particular locus across members of the genus Fragaria whereas FraCOL, FrvCOL, FrnCOL, and FriCOL are used to designate genes of F. ananassa F. vesca F. nubicola and F. iinumae respectively. Aside from numbering, genes were named for their apparent Arabidopsis homologs. Results Cloning And Identification Of Four CONSTANS-LIKE Genes Of Fragaria A series of EST libraries have been generated by this laboratory, representing transcripts accumulating in mature F. ananassa plants (Folta et al., 2005), developing flower buds, various root tissues, 12 fruit stages (K. Folta et al., unpublished) and F. vesca seedlings (J. Slovin et al., unpublished). These EST collections served as a basis for gene discovery based on direct sequencing, colony hybridization and PCR with degenerate primers. Simple homology comparisons to sequences within public databases identified convincing homologs of at least five distinct Constans -like ( COL ) genes. Four were designated FrCOL1 FrCOL2 FrCOL3, and FrCOL4 whereas the fifth, which shared the greatest homology with AtCO and the rice Hd1 was presumed to be the Fragaria ortholog of CO and designated FrCO These genes represent the canonical three distinct subgroups of the COL family as outlined by Griffiths et al. (2003) (Figure 3-1). Close matches to each of these genes were found among publicly available ESTs in other Rosaceae species as well (Table 3-1).

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47 A full-length cDNA clone of FraCO was obtained from a flower tissue library derived from F. ananassa ‘Strawberry Festival’. Seven individual clones were identified and sequenced, and all contained transcripts completely identical except that two contained a somewhat truncated version of the 3’ UTR. The 1,197 bp coding region shows considerable similarity to AtCO and other previously characterized homologs, with the most similar known transcript being PdCO1 from Populus (Table 3-1). Comparison of the amino acid sequence to those proteins designated as Group I suggested that FrCO belongs in Group Ia (Figure 3-2), a division that includes all genes experimentally demonstrated to complement the Arabidopsis co mutant and those believed most likely to play an important role in flowering behavior. The predicted protein contains a CCT domain and two intact B-Box regions, more closely resembling similar proteins in other eudicots, rather than the less conserved second B-Box present in some monocots (Martin et al., 2004). All four middle domains common to Group Ia COL proteins were also identified. Full-length coding sequences for three other FrCOL genes identified could be deduced from overlapping F. vesca ESTs, as well as part of a fourth. Although these genes contained the basic structures of COL genes, they bore less similarity to AtCO than did FrCO and appear unlikely to be the Fragaria ortholog of CO These were designated FrCOL1 FrCOL2 FrCOL3, and FrCOL4 FrCOL1 and FrCOL2 both encode predicted proteins consistent with Group Ic COL genes, with the major difference from the Group Ia genes being the presence of the M2c (Zobell et al., 2005) rather than the M2 domain (Figure 3-3). Several copies of both were identified among F. vesca EST sequences, as well as a single, slightly different, F. ananassa ‘Strawberry Festival’ FrCOL1 sequence. FrCOL1 is roughly equal in similarity to both AtCOL3 and AtCOL4

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48 at the amino acid level (55%), with AtCOL3 having slightly higher similarity in the major functional domains. It is most similar to MdCOL1 and MdCOL2 from apple, the only previously characterized COL genes in the Rosaceae (Jeong et al. 1999), and to PrpCO a COL gene from peach mapped by Silva et al. (2005). FrCOL2 while retaining a similar structure at the amino acid level, was not closely related to FrCOL1 at the nucleotide level, suggesting the two genes diverged some time ago. Among Arabidopsis COL genes, the predicted protein most closely resembled that of AtCOL5 Based on their relative frequency in BLAST-X searches of Rosaceae ESTs, FrCOL1 and its orthologs are possibly among the most commonly expressed COL genes in the family, whereas FrCOL2 and its homologs are expressed at a somewhat lesser level (data not shown). FrCOL3 was the least CO -like of the transcripts identified, with a structure consistent with a classification in Group II. FrCOL3 was found only in F. vesca libraries derived from young seedlings, and although matches were found among Rosaceae ESTs (primarily in an apple seedling library), it appears to be a much rarer transcript than the FrCOL1 and FrCOL2 genes. Only a partial sequence for FrvCOL4 was found among the sequenced ESTs, comprised of the B-Box region and much of the 5’ UTR. Although the full sequence was not available, this clearly represents a gene distinct from those previously described. The closest match in Arabidopsis was AtCOL10 indicating this is a likely member of Group III, and if so, all four groups present in Arabidopsis have also been identified in Fragaria (Figure 3-2). No members of Ib, Id, Ie, or IV were found, none of which have thus far been identified in eudicots (Griffiths et al., 2003). FrCO / FrCOL Gene Structure Group I and II COL genes in Arabidopsis possess a single intron located about a third of the length from the 3’ end of the coding region, between the M3 and M4 regions (Fig. 3-4).

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49 Using internal primers designed to include ~400 bp of the coding region and to flank the putative intron site (Table 3-2), based on the previously identified cDNA, partial genomic versions of FrCO, FrCOL1, and FrCOL2 were amplified and sequenced from genomic DNA of F. ananassa ‘Strawberry Festival’, F. vesca FDP815, and F. nubicola FDP601 (Figure 5-4.) Additionally, a product was also amplified in F. ananassa flower cDNA, using the same primer sets. In all cases, an intron was shown to be present at the predicted site in the amplified genomic product (Figure 3-4). FrCO intron length ranged from 704 to 746 bp. Introns in F. nubicola, F. vesca, and three nearly identical alleles from F. ananassa ( FaCO -35) were highly similar in both sequence and length (704, 707, and 706 bp, respectively), whereas a second F. ananassa allele ( FraCO-C9 ) was considerably different, containing a 28 bp insertion and numerous other polymorphisms, for a total length of 746 bp. Somewhat similar to this allele were those of F. iinumae and F. nubicola (Figure 3-5). Alleles also differed significantly in sequence at one site in the coding region, between the M1 and M2 domains. This region appears to be characterized by highly repetitive sequence containing multiple CAA codons, and is poorly conserved between and even within species (Figure 3-5A,B). FraCO-C9 and F. iinumae were considerably divergent in this region from the other alleles sequenced (Figure 3-5). FrCOL1 was shown to contain a 82 bp intron in both F. x ananassa and F. vesca similar in length to the related AtCOL3 (101 bp), but considerably smaller than that of AtCOL4 (403 bp). Additionally, a 12-bp insertion was identified in a single ‘Strawberry Festival’ EST, designated 7C. A primer landing in this insertion amplified a product in all eight octoploid cultivars tested, but not in any of the diploids, suggesting that this particular insertion may be unique to the octoploids.

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50 FrvCOL2 contained a 118 bp intron, whereas FrnCOL2 a 119 bp intron, both similar in length to the 88 bp intron of AtCOL5. The cDNA product for FrCOL1 was the same size as that predicted from the sequenced transcript. However two product sizes were observed for FrCOL2 consistent with the presence of an unspliced transcript variant, and a partial EST transcript containing nearly all of the intron sequence was identified among F. vesca sequence data. Southern blot analysis of FrCO Copy Number Southern blot analysis displayed a banding pattern consistent with a single FrCO locus in F. vesca as in other diploid organisms, showing two bands in those lanes where the probe fragment was cut once by the chosen enzyme, and one in those where it was not cut. The cultivated octoploid, F. ananassa however, showed multiple bands, frequently as many as six for enzymes that cut with in the probe region (Figure 3-7). Mapping Of Fragaria CO And COL Genes In A Diploid Mapping Population The fragment of FrnCO generated by the RN-FaCO primers was found to lack one of the two Hind III enzyme restriction sites possessed by the corresponding fragment of FrvCO and this was used to generate a CAPS marker for mapping (Figures 3-8A, 3-8B). This marker segregated in a 14:24:18 (aa:ab:bb) ratio in a subset of the reference population developed by Sargent et al. (2004), and was found to map to linkage group VI, with linkages to SSR markers EMFn153, UDF025, FAC-005, and EMFv160AD, with LOD values of 15.48, 7.79, 11.15, and 13.71 respectively (Figure 3-8C). No polymorphisms were found between F. vesca FDP815 and F. nubicola FDP601 in the intron and flanking coding region of FrCOL1 Two polymorphic restriction sites were identified in FrCOL2 and mapping was attempted first using EcoRV. Because of suspect segregation ratios and banding patterns that were difficult to resolve, a decision was made to score the marker as a dominant marker, but the incongruous segregation ratio persisted, yielding a 40:41

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51 (aa:b_) ratio. The first 24 individuals were also scored using the second enzyme, Afl III, yielding the same pattern seen among these plants with the first enzyme. The resulting mapping data did not convincingly place the gene on any of the seven linkage groups or in a strong linkage relationship with any of the established markers. Comparison Of Strawberry Genotypes Under Varying Photoperiods In order to test the expression of FrCO and other genes under various photoperiods, a pair of growth chamber experiments were performed. The first growth chamber experiment consisted of a comparison of the SD cultivar Strawberry Festival with the DN cultivar Diamante under 8 and 16 h photoperiods. After several weeks under these treatments, these genotypes reacted as predicted, with ‘Strawberry Festival’ flowering heavily under SD and producing numerous runners under LD, and ‘Diamante’ flowering vigorously under both photoperiods. The DN plants produced runners only under the 16 h photoperiod, and even then only produced a very few runners. Although observations were made, no flower or runner data were collected in this experiment. The second growth chamber experiment, under cooler temperatures, compared ‘Diamante’ to the SD cultivar Camarosa, and to a selection of F. vesca Hawaii-4. ‘Diamante’ again flowered under both conditions, more vigorously under LD than SD. ‘Camarosa’ did not runner and flowered only under SD. Unexpectedly, Hawaii-4, a yellow-fruited diploid selection presumed to be closely related to F. vesca ssp. semperflorens and previously classified as not photoperiod-sensitive (Oosumi et al., 2006), behaved in a clearly SD fashion, flowering profusely under an 8 h photoperiod and runnering primarily under LD (Table 3-4). The expression pattern of FraCO in the first study was studied by RNA-gel (northern) blot and subsequent hybridization to a radio-labeled CO probe. Transcript levels varied both between plants grown under SD and LD conditions, as well as between SD and DN genotypes

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52 (Figure 3-9). Under LD, transcript level in ‘Strawberry Festival’ peaks rather weakly before dawn, whereas under SD a strong peak appears in the early part of the day. Transcript levels in ‘Diamante’, however, are lower under both conditions, with a broader peak in the afternoon, later than in ‘Strawberry Festival’, and almost no transcript present under long days. One concern was that RNA-gel blot experiments may not give precise measurement of CO transcripts because of potential cross-hybridization with other family members. To verify the results, gene-specific primers were developed and used to evaluate steady-state RNA levels in a similar experiment, this time using semi-quantitative RT-PCR. Similar results were seen when the growth chamber experiment was repeated with ‘Diamante’, ‘Camarosa’, and Hawaii-4 (Figure 3-10) using these methods. All cDNA samples were tested with primers for ubiquitin and the ubiquitin control was used to balance template amount across samples within each set. Allele-Specific Expression Patterns Differ Betwen Short-Day And Day-Neutral Genotypes Although allele-specific primers for the FraCO allele types 35 and C9 amplified products from genomic DNA of ‘Strawberry Festival’ and ‘Diamante’, 33 cycles of RT-PCR showed that while both alleles are actively expressed in ‘Diamante’, only the FraCO-35 allele appears to be significantly expressed in ‘Festival’ at dawn under short days, although a trace of FraCO-C9 is visible. A more visible band appeared at 40 cycles, suggesting that trace amounts may still be expressed, although this may be the effect of buildup from slightly non-specific priming (Figure 3-11). Additionally, though the amount of FraCO-35 and FraCO-C9 product present at 33 cycles in ‘Diamante’ seems similar, the amount of FraCO-35 product present in ‘Strawberry Festival’ appears higher than either allele in ‘Diamante’. No differences were noted in the expression of the 7C allele of FraCOL1 which appeared to be actively and equally expressed in both cultivars (not shown). As before, template levels were adjusted by equalizing with ubiquitin controls.

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53 Expression Of Other Genes Under Different Photoperiods Expression of other COL genes in these samples was also been investigated using RNA from the preceding experiments to see if their expression correlated in some way with relevant phenomena. From the first experiment, cDNA was generated from RNA taken at the 5 AM time point (the point of peak FraCO expression under SD conditions, corresponding with hour 8 in Figure 3-9) and from the low point of FraCO expression, 5 PM, in the SD and LD ‘Strawberry Festival’ and ‘Diamante’ sample sets. These cDNA samples were used as template for RTPCR to compare quantitative differences in expression of the CO and COL genes, as well ubiquitin and actin as controls (Figure 3-12). The previously noted difference in FraCO level was observed again, and was accompanied by higher levels of FraCOL2 under short days, but FraCOL1 appears more highly expressed under long day conditions. No measurable levels of FraCOL3 transcript were observed under either condition in either cultivar. All transcripts except FraCOL1 were undetectable at the 5 pm time point under both SD and LD conditions, coinciding with the low point in FraCO expression seen in the previous experiment, suggesting that they too may cycle diurnally. Control genes were uniform between treatments, suggesting that the differences observed in specific targets were reflective of actual variations in transcript level. The same process was repeated for the entire diurnal time course of F. vesca Hawaii-4 from the second photoperiod experiment. In this case, very little expression of FrvCOL1 and FrvCOL3 were seen. FrCOL2 showed some evidence of circadian cycling, but not the large difference in amplitude between SD and LD seen with FrCO and in fact seemed to show evidence of a 12 hour period of variation (Figure 3-13).

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54 Some FraCOL2 Transcripts Contain An Unspliced Intron Under some conditions, a second product of higher molecular weight was visible for FraCOL2 The predicted size of this fragment was consistent with the retention of the 118-119 bp intron shown in the diploid FrCOL2 genes. Additionally, a partial FrvCOL2 EST, DY674801,was identified in Genbank with more than 95% of the intron, the sequence ending only a few bases short of the end. Conditions under which the unspliced transcript was detected varied between genotypes. In ‘Strawberry Festival’, it was present only under LD conditions, at the 5 am time point, whereas in ‘Diamante’ it was visible only under SD conditions, but at both 5 am and 5 pm (not shown). In F. vesca in the second trial, however, it was clearly visible at two points of the day under short photoperiods (Figure 3-12). Discussion The Co Gene Family In Fragaria (Rosaceae) The results of these studies clearly demonstrate the presence of a family of COL proteins in Fragaria similar to those previously demonstrated in a number of other species, accounting for all subgroups demonstrated in Arabidopsis and including a likely homolog of CO The five members of the COL family identified in this work cover the full range of COL diversity described in the Arabidopsis, the only eudicot in which the entire family is known to be documented, with representatives in all subgroups. Unlike in Arabidopsis, only a single member of Ia, FrCO was identified. Unless there is significant deviation at the nucleotide level, the possibility of other Ia COL genes in Fragaria seems remote. Both the results of the F. vesca Southern blot, and the fact that no others were isolated in two hybridization experiments, one probed with AtCO and the other with FraCO suggest that other such genes may not exist in strawberry. No evidence of multiple group Ia loci was seen among ESTs from other diploid Rosaceae species, although two distinct but closely-related classes of CO were found in apple.

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55 Multiple group Ia genes are common, even in diploid species, however, and have been demonstrated in poplar, tomato ( Lycopersicon esculentum Mill.), barley, and oilseed rape ( Brassica napus L.) as well as in Arabidopsis (Fig. 3-1), but have been shown not to be the case in rice (Yuceer et al., 2002; Ben-Naim et al. 2006; Griffiths et al., 2003; Robert et al., 1998). It is possible that rather than possessing distinct roles, the nonCO group Ia genes in these species are merely the result of duplication, coupled perhaps with at least partial loss of function within the genome, although AtCOL1 has been shown to shorten the circadian cycle when overexpressed in wild type plants (Ledger et al. 2001). AtCO and AtCOL1 are very similar, even at the nucleotide level, but AtCO overexpressors show no change in circadian cycle. However it is possible that reciprocal mutations have separated functions of the original gene into the two genes, with the current AtCO retaining the ability regulate flowering and AtCOL1 possessing a role in circadian regulation. Although FrCO mapped to the same linkage group in the diploid population as the seasonal flowering locus ( SFL ), there was no significant linkage with this locus, as 38cM separate the two, casting doubt on the idea that non-seasonal F. vesca may be FrCO mutants. Neither of the two QTLs for date of first flower in the diploid mapping population mapped to the location of FrCO (Sargent et al., 2006a). This suggests that variation in FrCO is not a significant source of variation in flowering time in this population, and that unlike in other species, such as rice (Yano et al., 2002), the lack of seasonality is not the result of mutations in the CO gene. Homologs of all Fragaria COL genes were found in ESTs from other Rosaceae species, though in no case were homologs of all five genes found in a single species (Table 3-3). This is most likely due to the limited number ESTs available from a given species, as COL proteins are

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56 generally relatively low in abundance and only apple begins to approach the large numbers of ESTs need to reliably locate rare transcripts. Searches of publicly availabe ESTs identified group Ia genes in both Malus and Prunus species, and it appears that there may be two distinct members of the group in apple. Unlike strawberry, apple does not appear to be photoperiodic (Carew and Battey, 2005). However, group Ia COL genes nonetheless appear to be present in all higher plant species thus far investigated, including some that lack photoperiodic sensitivity, such as tomato ( Lycopersicon esculentum ) (Ben-Naim et al. 2006). Group Ic, homologs of AtCOL3/4/5 represented in Fragaria by FrCOL1 and FrCOL2 appear to be among the most highly expressed COL genes in the Rosaceae, constituting 6 of 19 COL ESTs identified in Fragaria 13 of 20 in Prunus and 87 of 106 in Malus, of which a large majority are homologs of FrCOL1 Prior to this study, the only Rosaceous genes yet characterized were members of this group, a single pair from Malus domestica MdCOL1 and MdCOL2 (Jeong et al, 1999). A very similar gene designated PrpCO was also mapped to Prunus linkage group G1 on P. dulcis P. persica F2 map by Silva et al. (2005), but was not further characterized. The group Ic genes may be the most ancient members of the family, being the only COL genes identified in the primitive plant P. patens (Zobell et al. 2005). As with all nonCO members of the family, the function of these genes is still unclear. Zobell (2006) found no discernible phenotype in single mutant knockouts for any of the three members in P. patens However, work by Datta et al. (2006) has shown early flowering and changes in lateral branching and root development in Arabidopsis COL3 knockout mutants, and have suggested a

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57 role as a positive regulator of red light signaling. FrCOL1 and its homologs may have similar functions in the Rosaceae. Although two polymorphisms suitable for mapping were detected, mapping FrCOL2 in the diploid reference population proved difficult, and it was not possible to identify linkages with any published markers. Scored as a dominant marker, one would anticipate a segregation ratio of 3:1, but in this case the pattern was close to 1:1, skewing towards the F. vesca parent. Some parts of this map have shown considerable distortion (Sargent et al., 2004a, 2006b), including one marker, BFACT-010, that segregated 79:5 rather than 3:1 (Sargent et al., 2006b). This distortion of segregation ratios may be due to the parents chosen as parents of the reference population. Although Sargent et al. (2004a) produced their population from two selfed F1 F. vesca x F. nubicola hybrids, previous researchers found F1 plants to be self-incompatible, like the F. nubicola parent (Evans and Jones 1967) or sterile (Dowrick and Williams, 1959). Although the mechanics of self-incompatibility have yet to be elucidated in strawberry, it may be that this system remains functional to an extent, resulting in bias in what pollen may successfully fertilize the F1 plants. Additionally, although sexually compatible, F. vesca and F. nubicola do not overlap in their ranges and are distinct on both a morphological and molecular level (Sargent et al. 2005, 2006b; Potter et al., 2000) suggesting that there has been time for divergence between these species. Such divergence might result in imperfect pairing of chromosomes, and if largescale rearrangements exist between the species, may even be reflected in the formation of multivalents and gametes with chromosomal abnormalities. FrCOL3 is the sole representative of group II COL genes identified. While maintaining the general COL structure, group II genes have only one B-Box and the four conserved middle regions. Though common to both monocots and dicots, comprising 4 of the 17 Arabidopsis COL

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58 genes and 3 of the 16 in rice, their function remains unknown, and this may represent the first report of such a gene outside of these two model systems. Apparent homologs of FrCOL3 were identified among ESTs of only one other Rosaceae species, peach, although a distinct transcript matching the criteria of a group II COL gene was found among apple ESTs as well. Mining of EST libraries revealed only one partial transcript of FrvCOL4 consisting of the 5’ end the gene. Although we can only guess at the rest of the gene’s structure, this end is clearly characteristic of a class of COL gene not otherwise represented by the other FrCOL genes, group III. The B-Box region of group III genes is distinctive because instead of a pair of B-Boxes, these genes have a single B-Box followed by a modified zinc-finger region. Although impossible to observe in an EST, these genes are also distinguished from the others in the family because of their differing intron structure, with two splice locations in the middle of the gene and a third within the CCT region, none of which correspond to the intron location in the other family members (Griffiths et al. 2003). This suggests that the differentiation from the other COL genes is very ancient. The function of this class of genes is still imperfectly known, but AtCOL9 has been shown to play a role in flowering, apparently through the suppression of CO and, as a result, FT (Cheng & Wang, 2005). A related gene, AtCOL10 was also shown to have similar role in early work presented by the same authors (Cheng & Wang, 2006), and both appear to be expressed in a diurnal fashion. Evolutionary Relationships Among FrCO Sequences The relationships between the octoploid FrCO alleles and those of the diploid species reinforce some aspects of the established views of the origins of the diploid genomes of F. ananassa while perhaps casting doubt on others.

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59 One allele class, FraCO-35, bears striking resemblance to that of F. vesca Within the coding region, only the previously mentioned variable site between the M1 and M2 regions differs, and in fact this region differs considerably even between two selections of F. vesca Hawaii-4 and FDP815. Even within the intron, the nucleotide sequence is 98% identical between FraCO-35 and F. vesca (Figure 3-5). The idea that F. vesca may represent a diploid progenitor of the octoploids is an old one, suggested first by Ichijima (1926) and reinforced by further work, both cytogenetic (Senanayake and Bringhurst, 1967) and molecular (Potter et al, 2000; Folta and Davis, 2006; also Chapter 5 of this dissertation) and this result seems to strengthen this view. The origin of the other component genomes has never been as clear, though a number of species have been suggested. The sequence of the other type of octoploid allele, FraCO-C9 does not seem to clearly implicate any of these, but may indirectly point to two species, F. nubicola and F. iinumae F. nubicola has long been discussed as a progenitor of the octoploid species, and was suggested as a possible source of the A or A genomes by both Senanayake and Bringhurst (1967) and Potter et al. (2000). Staudt (1989) had proposed F. nubicola along with F. vesca as the ancestors of the hexaploid species F. moschata but sequence data analyzed by Potter et al. (2000) implicates F. orientalis and Lin and Davis (2000) showed that F. viridis was the likely cytoplasm donor for F. moschata The range of F. nubicola, today confined to a relatively small area of South Asia, does not overlap the ranges of F. moschata, F. viridis or F. vesca. If F. nubicola is in fact an ancestor of both F. moschata and the octoploid species, this may suggest that it or a closely related species once existed across a much broader geographic area.

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60 F. iinumae has primarily been implicated in unpublished work by DiMeglio and Davis (cited in Folta and Davis, 2006). Currently, populations of F. iinumae are confined primarily to the island of Hokkaido and the Kuril Islands. The species appears to be an outlier from the rest of the genus both at the morphological level (Sargent et al., 2004b), and at the molecular level (Harrison et al, 1997; Potter et al., 2000, Sargent, 2005). F. iinumae has proven incapable of producing hybrids with other diploid species (Bors, 2000; Sargent et al., 2004b) but polyploids derived from the species do not appear to have been investigated and may in fact be capable of producing allopolyploid offspring in combination with other species. No diploid species contained the 28bp insertion in the intron of the FraCO-C9 allele, and F. iinumae contained 5bp and 7bp intron deletions not seen in FraCO -C9 or any other allele. However, an inspection of SNPs within the intron on either side of the 28 bp insertion reveals an interesting pattern—on the upstream side of the insertion, C9 and F. nubicola share the same base at 7 of 8 SNP sites, compared to only 1 between C9 and F. iinumae Downstream of this insertion, however, more SNPs match F. iinuame with 9 of 18, compared to 6 matches between C9 and nubicola and 3 sites where C9 did not match either species. Small microsatellite repeats in the intron, all downstream of the insertion, also seem to more closely resemble F. iinumae with two of the three identical between the two alleles (though the difference between F. nubicola and the other two alleles is only one repeat in both cases), and the third SSR containing five repeats of AT in FraCO-C9 four in F. iinumae and three in all other sequenced alleles. The coding regions of the gene are nearly identical among all alleles, with the exception of the variable region between the M1 and M2 domains, which matched exactly between FraCO-C9 and F. nubicola Given the level of polymorphism in this region, with even members of the same

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61 species showing variability, this evidence would seem to make a strong case that at least this portion of the gene originated in F. nubicola The division seemingly indicated by the 28 bp insertion may represent a slightly uneven crossing over event in the species’ past, creating a chimeric gene with an upstream end derived from F. nubicola or a near relative, and a downstream end derived from F. iinumae or an ancient related species. If this locus lies in a region characterized by frequent crossover or disruption by interchanges between genomes, this may explain the failure to express of the FraCO-C9 allele seen in ‘Strawberry Festival’, for example because of damage to the promoter. Nemoto et al. (2003) found that the CO locus in one of the three genome pairs of hexaploid was not expressed, despite an intact coding region, and traced this to an 63 bp deletion in the promoter upstream from the gene. It seems unlikely that these two classes represent the entire octoploid complement of FraCO allele types. Only FraCO-35 -type sequences were identified in sequencing of cDNA from ‘Strawberry Festival’, and only two identical FraCO-C9 sequences were identified (by the size difference caused by the insertion and by failure to cut with Xba I) among 16 clones amplified from genomic DNA of ‘Strawberry Festival’. It is possible that alleles are not being identified by the PCR-based methods used in this study due to mismatches in primer sequence, or that the FrCO locus has been eliminated from one or more of the component genomes of the octoploid. FrCO Expression In previously characterized SD plants, such as rice (Yano et al., 2000) and perennial ryegrass (Martin et al., 2004), CO expression patterns remained about the same as that seen in Arabidopsis. The differences in flowering behavior come as a result not of differences in CO

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62 expression pattern, but from differences in the output, with CO acting as an inhibitor of FT and SOC1, and hence flowering, rather than promoting them as in Arabidopsis. In strawberry, it appears that something quite different is occurring. Under short days, FrCO transcript levels conform to a robust diurnal cycle, though the peak is shifted so that unlike under SD in Arabidopsis, it occurs during the day, rather than after dusk. Under long days, however, there is little FrCO expression at all, though there is a perceptible peak during daylight hours as well. Based on transcript accumulation alone, this suggests that FrCO like AtCO is a promoter of flowering, and that the lack of flowering under long days does not necessarily derive from an inhibition of flowering, but instead from a lack of promotion, and possibly inhibition through other mechanisms. Interestingly, even the DN cultivar, Diamante, displayed this pattern, suggesting that the critical difference between SD and DN cultivars is not one of CO expression. If so, then flowering under LD conditions in Diamante must be the result of elements further down the pathway than FrCO. Expression of FrCO in ‘Diamante’ was slightly different than the other cultivars, with a somewhat flatter, wider peak. In light of the evidence that one of the two FraCO alleles in ‘Strawberry Festival’ is being expressed at greatly reduced levels, while ‘Diamante’ expresses both alleles robustly, this broad peak may be the result of two or more alleles expressed somewhat out of phase with each other, with the overlapping curves giving the impression of a wider peak. Some caution is warranted in the interpretation of these results, as this data reflects only mRNA transcript levels. Analysis of transcript levels does not take into consideration the mechanisms that regulate post-translational regulatory mechanisms. Elegant studies by Valverde et al. (2004) demonstrate that light-quality driven alterations in CO stability and localization are critical components of CO action.

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63 The expression pattern seen in strawberry does not appear to have been previously described, and may represent a fundamental change in the perceived function of CO In Arabidopsis and rice, according to the external coincidence model, flowers are initiated or inhibited in response to whether the peak of CO occurs in daylight or darkness. Without knowing the dynamics of protein level, it is difficult to say for certain what the mechanism is here, but it appears that the critical factor is not one of when the peak in CO occurs, but how high it is. Whether this trend seen at the transcript level is continued at the protein level, where further regulation may occur (Valverde et al., 2004), remains to be seen. If it is, then some factor other than the integration of photoperiod and circadian cycle provided by CO is likely responsible for the perception of photoperiod in strawberry, at least with respect to flowering. CO transcription is regulated by a number of factors, and a switch between SD and LD flowering in eudicots may be a result of a change in one of these elements, rather than a change in the functioning of CO itself. The evidence available does not seem to clearly indicate a mechanism for the reduced FrCO expression seen under LD, but several mechanisms that either reduce CO transcription or encourage degradation of the transcript have been identified. A probable blue-light photoreceptor, FKF1, appears to regulate CO transcript level by repressing CDF1, a repressor of CO transcription (Imaizumi et al., 2003). Other possibilities include the Group III COL proteins, as both AtCOL9 (Cheng and Wang, 2005) and AtCOL10 (Cheng and Wang, 2006) have been shown to decrease CO transcript level in Arabidopsis overexpressing those genes. The new evidence also calls into question the role of phytochrome in the regulation of flowering, and the degree to which it is maintained across species. In Arabidopsis and other LD plants, far red-enriched extensions of day length at the end of the day accelerate flowering,

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64 whereas red light extensions delay flowering (Kadman-Zahavi and Ephrat, 1974). This has been explained by Valverde et al. (2004), who found that phytochromes mediated the stability of the CO protein, with phyB, activated under red light, encouraging the degradation of the protein in the morning, and phyA, activated under far-red, stabilizing the protein. The phyB receptor also regulates flowering through the upregulation of PFT1, a repressor of FT (Halliday et al., 2003) as well as possibly promoting the expression of LFY (Blzquez and Weigel, 1999). While this model explains the flowering responses to light quality observed in LD plants, it becomes clear that something different is happening in SD plants. Vince-Prue and Guttridge (1973) found that just the opposite occurred in strawberry, with far red extensions inhibiting flowering if applied at the beginning of the day, and red light extensions inhibiting if applied at the end of the day. Kadman-Zahavi and Ephrat (1974) grew plants under blue light supplemented with far red, and found flowering delayed compared to blue light alone or shaded conditions. While this might seem consistent if FrCO was a repressor of flowering, the expression data seen in our study would seem to suggest that it is not. In fact, rice, in which CO actually has been determined to be a repressor of flowering, does not display this pattern, but instead reacts in the same manner as LD plants (Kadman-Zahavi et al, 1976). Because most of this regulation is occurring at the level of the protein, it is difficult to gauge what is occurring in strawberry based solely on our transcript level data. Kadman-Zahavi and Ephrat (1974) described two clear groups of SD plants, based on this reponse. With strawberry, they place Chrysanthemum morifolium Ramat. and P. nil while in the group of SD plants that respond like LD plants to red and far-red light they place Mimosa pudica L Amaranthus sp., and Cosmos bipinnatus Cav.. Descriptions from earlier work also suggest that Salvia occidentalis Sw. is also similar to strawberry (Meijer, 1959), whereas corn ( Zea mays

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65 L ) rice and sorghum ( Sorghum vulgare L.) belong to the other group (Kadman-Zahavi et al, 1976; Lane, 1962). Roses, close relatives of strawberries, have been shown to have an increase in flowering in response to red light extensions at the end of the day (Maas and Bakx, 1995, 1998), so it appears both groups may exist even within the Roisoideae. If so, this may imply a relatively simple difference in the mechanisms controling such responses. Expression of Other COL Genes Despite belonging to a common subgroup, FraCOL1 and FraCOL2 displayed rather different expression profiles. FraCOL2 appears to be expressed in a pattern similar to FraCO a pattern previously shown to be common to other COL group Ic genes in Arabidopsis. Microarray data by Smith et al. (2004) show that AtCOL3 and AtCOL4 follow similar diurnal patterns, with peaks around dawn under long days. Most other COL genes that have been investigated, including AtCOL1 AtCOL2, and AtCOL9 (Ledger et al. 2001, Cheng & Wang, 2005) also show this pattern. The variation in splicing efficiency of FraCOL2 does not seem to follow a clear pattern, however it may serve as a means of negative regulation of its function, as the introns in F. nubicola FDP601, F. vesca FDP815, and F. vesca Hawaii 4 all contain multiple stop codons that would result in a truncated protein. Such a truncated protein would lack the M4 and CCT domains. Little is known about the functions of these elements. The CCT domain has been proposed to be involved in the localization of the protein; however the co mutants co-5 and co-7 with mutations in the CCT domain, localize correctly but do not function properly (Robson et al., 2001). Given that the function of FrCOL2 is unknown, it is difficult to speculate on the role of this truncated protein, if any, but it seems conceivable that it might compete with the functional version of the protein in its interactions with either DNA or protein.

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66 Splice variants of the CO ortholog PnCO of Japanese morning glory were documented by Liu et al. (2001), who noted both unspliced and alternatively spliced transcripts in addition to the correctly spliced version. A majority of transcripts contained the full intron, whereas smaller numbers were properly spliced or retained a small 26 bp segment of intron sequence. The partial retention of the intron resulted in a frameshift and premature stop codon, but the presence of the entire intron caused the coding sequence to remain in the correct reading frame. Despite this, only the properly spliced transcript was able to complement Arabidopsis co mutation in transgenic plants. The flowering-related gene Proliferating Inflorescence Meristem (PIM) from pea, a homolog of the Arabidopsis meristem identity gene AP1, has also been shown to commonly exist as an unspliced transcript (Taylor et al., 2002). Transcripts with unspliced introns are moderately common in plants (Ner-Gaon et al., 2004). Alexandrov et al. (2006) found that about 7% of transcripts demonstrated some sort of alternative splicing, of which 27% (Alexandrov et al., 2006) to 38% (Iida et al., 2004) contain one or more unspliced introns. Ner-Gaon et al. (2004) found that transcripts associated with physiological flux were more prone to intron retention than those involved in functions such as metabolism and housekeeping. Iida et al. (2004) found that retained introns were more common in plants that have received a recent stress, such as cold, heat, ultraviolet light, dehydration, and various hormone treatments. By producing non-functional truncated products or, alternatively, products that are degraded by nonsense-mediated decay (NMD) caused by frameshifts (NarGaon et al., 2004), an extra level of regulation in genes that are critically sensitive to changes in transcript level may be provided.

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67 Unlike FraCO and FraCOL2, FraCOL1 transcript appears to remain at relatively constant levels through the course of the day. It is the only FraCOL gene expressed in all cultivars at the 5 pm time point under short days, the apparent low point of the FraCO cycle. In contrast, the closest Arabidopsis homologs, AtCOL3 and AtCOL4 are both expressed in a diurnal fashion (Smith et al., 2002), although Jeong et al. (1999) did not investigate diurnal expression of the apple homologs of the FraCOL1 gene that they characterized, nor is it clear at what point or points in the diurnal cycle tissue was collected. While both genes were expressed at the same relative level in all tissues, MdCOL2 was consistently expressed at a higher levels than MdCOL1 And while most tissues showed a uniform but low level of expression, higher expression of both genes was seen in developing fruit and flower tissue, which may partly explain the great number identified in apple and peach EST libraries, because many of these were derived from fruit tissue. In strawberry, however, the majority of ESTs are from libraries derived from developing seedlings not yet producing flowers or fruit, so bias in tissue of origin cannot entirely explain the preponderance of this group among Rosaceae ESTs in GenBank. Datta et al. (2006) oberseved decreases in the lateral branching of inflorescences in Atcol3 mutants, and found evidence that AtCOL3 promotes the formation of branches and inhibits the growth of the primary shoot during short days. If FraCOL1 has a similar function in strawberry, it may be that selection for increased numbers of inflorescences or proliferation of branch crowns has selected for a mutation that results in continual expression of this gene, rather than diurnal fluctuation. Similarly, it is also possible that the elevated expression levels for FraCOL1 and the two Malus genes seen in fruit and flower tissue derives from a role within infloresence development, and that selection for increased flower number might have encouraged selection for relatively

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68 continuous expression. It would be interesting to examine the expression of this gene in F. daltoniana a strawberry species characterized by single flowers (Sargent et al. 2004b). Datta et al. (2006) demonstrated the necessity of a pair of amino-acids, VP, near the C-terminal, for binding with COP1 and conferring normal red-light phenotype. FrCO, FrCOL1, and FrCOL2 all possess this motif, but it is lacking in FrCOL3 (the C-terminus of FrCOL4 is currently uncharacterized, although group III genes in rice and Arabidopsis lack this pair), suggesting possible roles for these first three, at least, in photomorphogenic development. FraCOL3 was not expressed at perceptible levels in any of the RT-PCR experiments, though we were able to amplify a weak band from ‘Strawberry Festival’ flower cDNA. FrvCOL3 occurs several times among Fragaria EST sequences, and it is worth noting that all of these are from developing seedlings, although there are only a relatively small number of ESTs available from mature plants. In Malus and Prunus FrCOL3 -type transcripts occur several times in mature and seedling material. Two Arabidopsis orthologs, AtCOL6 and AtCOL7 coincide with QTLs for rosette leaves at flowering, days to budding, and days to flowering, suggesting possible roles in flowering, although they share this interval of the chromosome with other flowering and development related genes such as MADS-Box transcription factors and genes involved in meristem development and hormone synthesis (Bandaranayake et al., 2004). Materials and Methods Plant Material Six octoploid cultivars of F. ananassa were used during the course of the work. These cultivars and the sources from which they were obtained are given in Table 3-5. All were obtained as rooted plants but were propagated as needed throughout the course of the experiments. Only the second group of ‘Diamante’ was used directly from the nursery; all others were multipled by runners in the growth chamber prior to experiments. F. vesca Hawaii-4 was

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69 obtained as runner plants from the collection of Dr. Thomas Davis at the University of New Hampshire and propagated by runners in the laboratory for use in the experiments. Plant Growth Conditions Plants were maintained on shelves in the laboratory at room temperature (23C), under cool white fluorescent lighting when not in use in diurnal experiments. The first diurnal experiment, utilizing ‘Strawberry Festival’ and ‘Diamante’, was conducted in the growth chamber in enclosed compartments, under either 8 h or 16 h photoperiods under cool white fluorescent lighting. Each treatment consisted of three 25 cm pots per genotype, containing four mature plants in ProMix BX soilless medium, and the pots randomized within the compartment. Temperature was maintained at approximately 23C. The second diurnal experiment was conducted in growth chambers, with six plants in each treatment of the octoploid cultivars Earliglow and Diamante, and two each of Camarosa. Ten plants of F. vesca Hawaii-4 were also grown in each chamber. Plants were grown in 10-cm square pots in ProMix BX soilless medium and watered and fertilized as needed. Once again, 8 h and 16 h photoperiods were used, with a photon flux of approximately 300 mol•s-1•m-2 at the level of the soil surface. Photon flux was found to be fairly uniform through the chamber. Three flats containing two of each F. x ananassa cultivar were arranged in the center of the chamber, and the arrangements of the pots within each flat randomized. F. vesca plants were placed together in a fourth flat. Temperature was set at 18C day / 16C night. Plants in both experiments were watered and fertilized as needed. Total number of runners and inflorescences were counted on each plant at 4, 6, and 8 weeks from the beginning of the light treatments, except for the F. vesca for which data was collected only at 6 and 8 weeks.

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70 Cloning of FraCO A partial EST sequence (CO380854) resembling CO was identified among sequences derived from the octoploid ‘Queen Elisa’. A single primer based on this sequence, FaCOtop (5’TGGATGTTGGAGTTGTACCAG-3’) was used along with the M13 reverse primer to amplify a product from a mixed tissue ‘Strawberry Festival’ cDNA library by PCR. The product generated was cloned and sequenced, and a second primer, FaCO-R (5’CGGCATTGTTCCTTCATACTAA-3’ ) was developed. FaCOtop and FaCO-R were used to amplify a 344 bp product for use as a probe, using Touchdown PCR as described in Sargent et al. (2004). This probe was hybridized against approximately 20,000 colonies of a F. x ananassa ‘Strawberry Festival’ flower tissue library in E. coli / Gateway vector. Identification of COL Genes in Genebank One Fragaria COL gene, FraCOL1 had previously been identified in the description of an earlier EST library (Folta et al., 2005). All others were identified by TBLAST-N searches (Altschul et al., 1997) of Fragaria ESTs in GeneBank, using each Arabidopsis COL gene as the query, as well as searches using only AtCO CCT domain and B-Box region. Identified Fragaria sequences were also searched against Malus Prunus and Rosa ESTs to identify other Rosaceae homologs. Gene Structure Characterization Primers flanking the full coding region for each of the FraCOL genes were designed, as well as a primer set (RN) enclosing a roughly 400 bp segment of the coding region believed to include the putative intron site (Table 3-2). This segment of each gene was amplified from genomic sequence using Touchdown PCR (as Sargent et al. 2004, except extension time was increased to 1 min) from ‘Strawberry Festival’, FDP601, and FDP815, and from ‘Strawberry Festival’ flower cDNA. The presence of intron sequence was verified by comparing the fragment

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71 size generated from genomic sequence to that generated from cDNA and the size predicted from the EST or ESTs that it was identified from. Combinations of the flanking and RN primers were used to confirm the absence of other introns. Southern Blot Analysis Total DNA was extracted from of F. ananassa ‘Strawberry Festival’ and F. vesca Hawaii-4 using a modified cold CTAB method (Tombolato et al., in preparation). Ten g of each were digested with four restriction enzymes: Eco RV, Hind III, Bam HI, and Eco RI. Digested samples were separated by size using gel electrophoresis on 0.8% agarose gels, blotted onto nylon membranes, and UV cross-linked. A FraCO probe was developed using the RNFrCOF and FrCOfull R (Table 3-2) with ‘Strawberry Festival’ genomic DNA in a touchdown PCR reaction as described above. The probe was labeled by random priming as per manufacturer’s instructions, hybridized overnight at 60C, and given three washes of 20 minutes each with with a wash buffer comprised of 1x SSC and 0.1% SDS. Genetic Linkage Mapping COL genes in Fragaria FrCO was mapped in a diploid reference population developed by Sargent et al. (2004, 2006). This is a F2 population from the cross of F. vesca FDP815 x F. nubicola FDP601, and consists of 94 individuals. The RN-FrCO primer set (Table 3-2) was used to amplify a product by PCR from each individuals DNA (30 cycles, 52C annealing, 1 min extension). Ten l of each PCR product was digested in a reaction with 0.3 l of Hind III enzyme, 1.3 l 10x bovine serum albumin, 1.3 l 10x Promega Buffer B, and 0.3 l dH2O. Digested product was separated by gel electrophoresis on 1% agarose gels, stained with ethidium bromide, and photographed under UV light. FDP601 alleles displayed three bands when digested, whereas FDP815 displayed only two. Size differences allowed the trait to be scored as a co-dominant marker.

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72 Attempts to map the FrCOL2 locus were performed as above, with the following exceptions: the RN-FrCOL2 primer pair (Table 3-2) was used, extension time was set at 45 s per cycle, and PCR products were digested with Afl III enzyme. When this showed an unusual segregation pattern, the process was repeated with the Eco RV enzyme. In both cases, the FDP815 allele was not cut by the enzyme, whereas the FDP601 allele was. However, due to incomplete digestion, it was scored as a dominant marker. Marker data was entered into the JoinMap 3.0 software program and mapped relative to marker data provided by Dan Sargent, consisting of most of the markers appearing in Sargent et al., 2004 and Sargent et al., 2006. Extraction of Nucleic Acids Except as noted above for use with the Southern blot, DNA was extracted using the Qiagen DNEasy Plant DNA extraction kit, according to the manufacturer’s instructions. Some genomic DNA was provided by others, namely that of F. vesca Hawaii-4, F. iinumae FRA377 F. bucharica FRA520, and F. mandshurica FME, from Dr. Thomas Davis at the University of New Hampshire, and F. vesca FDP815 and F. nubicola FDP601 and that of the F2 mapping population from these parents from Dr. Daniel Sargent of East Malling Research. RNA was extracted using a modification of the pine cone method of Chang et al. (1993) as described in Folta et al. (2005). Briefly, 1 g of tissue was frozen with liquid nitrogen and ground with a mortar and pestle, then incubated at 65C for 10 min in a CTAB-based extraction buffer (2% CTAB, 2% polyvinylpyrrolidone, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl, 0.5 g/ml spermidine, and 2.0% beta-mercaptoethanol). Samples were then allowed to cool to room temperature and equal volumes of chloroform:octanol were added and the samples homogenized using a Polytron T10-35 tissue homogenizer at 90% of full speed. The organic and aqueous phases were separated via centrifugation at 8,000 x g. The supernatant was then

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73 removed, mixed with equal volumes chloroform:octanol, and then separated via centrifuge again. LiCl was added to the resulting supernatant to a concentration of 2.5 M and allowed to precipitate overnight at 4C, after which it was centrifuged again at 10,000 x g. The pellet was resuspended in 500 l SSTE (1 M NaCl, 0.5% SDS, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA) and again purified with chloroform:octanol, then precipitated with two volumes of 100% ethanol. The pellet was then washed with 76% ethanol, 0.3M sodium acetate, dried using a SpeedVac, and resuspended in 50 l of 10 mM Tris-HCL (ph 8.0), 2.5 mM EDTA. Samples were quantified by spectrophotometry. Reverse Transcription and RTPCR For the first photoperiod experiment 1 g of RNA from each condition was reverse transcribed using AMV reverse transcriptase (Promega Inc., Madison, WI) as per the manufacturer’s protocol. The resulting cDNA was diluted 1:10 with TE buffer and used as template for RT-PCR reactions. Initial experiments were conducted with the ‘Camarosa’ SD cDNA set to determine the likely linear range for each pair of primers. For RN-FrCO, RN-Ubiq, and RN-Actin primers sets, reactions were conducted at 24, 28, 32, and 36 cycles, and the highest number of cycles which retained the relative band intensities seen at lower numbers of cycles was selected. RT-PCR reactions used the RN primer sets described in Table 3-2 using standard 3 step PCR (52C annealing temperature, 30 seconds extension, for 32 cycles for RN-FrCO and RN-Actin, 28 cycles for RN-Ubiq), separated by electrophoresis on 1% agarose gel, then stained with ethidium bromide and photographed under UV. Template amounts were adjusted using ubiquitin and actin RN primer sets (Table 3-2) to assure similar amounts of template DNA between cultivars and treatments and the PCR repeated and gels photographed (Figure 3-13). Allele-specific RT-PCR trials used the same PCR conditions, but 33 cycles was used because slightly less product was

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74 anticipated than for with the RN-FrCO primer pair. A second round was conducted at 40 cycles, with the idea of reach saturation and testing the whether any FrCO-C9 product was present in ‘Strawberry Festival’. The second experiment used a similar procedure, but reverse transcription was done using ImProm II reverse transcriptase (Promega Inc., Madison, WI) as per manufacturer’s instructions. The resulting 20 l reaction was not diluted but used directly as template in a PCR reaction identical to above, except that 35 cycles was used for FrCOL2 Template volume used in each reaction was adjusted using ubiquitin and actin RN primers to equalize template amounts. The products were again separated by electrophoresis (on a 1.5% agarose gel), stained and photographed.

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75 Table 3-1. Comparison of amino acid identity (%) of the predicted protein encoded by FraCO to those of Group Ia CONSTANS-LIKE genes in apple ( Malus domestica ), castor bean ( Ricinus communis ), Cottonwood ( Populus deltoides ), thale cress ( Arabidopsis thaliana ) rape ( Brassica napus ), Japanese morning glory ( Pharbitis nil ), rice ( Oryza sativa ), wheat ( Triticum aestivum ), and perennial ryegrass ( Lolium perenne ).Gene Acc. No.B-Box 1B-Box 2M1M2M3M4CCTOverall MdCOZ95979252801009775 RcCOy83929247701009771 PdCO1 AY51515086100926890809369 AtCO NM1215897682606360608854 PnCO AF3007007482784755809756 BnCOa1 AY2808687474455770308850 OsHd1 AB0418407680425850608850 TaHd1-1 AB0944906775332250508843 LpCO AY5532976751584750508441 Z Malus domestica CO gene assembled from ESTs EB130204, EB141442, and DR996733y Ricinus communis CO gene assembled from ESTs EG683156 and EG686997

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76 Table 3-2. RT-PCR primers for strawberry CONSTANS-like genes and controls, sequence source, TM, and approximate size in cDNA (as calculated from sequence).Gene PrimersSequence TM (C) ESTbp CONSTANS-like Genes FrCO RN-FrCO-F RN-FrCO-R ctgaatcctgtgaagaacagc tggatgttggagttgtaccag 53.6 54.4 —z388 FrCO-35 RN-FrCO-35-Fcatctgatcagaaccagttca52.1— z265 FrCO-C9 RN-FrCO-C9-Fcatctgatcagaacctcg55.3— z256 FrCOL1 FrCOL-7C RN-FrCOL1-F RN-FrCOL1-R RN-FraCOL7C-F ctgaggccgaggctgcttcg ctgtaccttaacaccctggc gatggcaacatgctgacggac 63.2 55.3 59.0 CX661699 DY674862 CO817106 403 117 FrCOL2 RN-FrCOL2-F RN-FrCOL2-R tgatgccctcgacatgaagc cccggtccactccggtcagc 57.6 65.1 DY669887 DY671835 411 (531)yFrCOL3 RN-FrCOL3-F RN-FrCOL3-R ttccaacgctgtttccaacg ctcagcctccatgagcaccgc 56.3 62.9 DY667001 DY673134 418 Control Genes FrActin1 RN-Actin-F RN-Actin-R tggctgtgcacgatgattgc taacttcccaccagatatcc 58.8 50.9 DV439971430 FrUbiq1 RN-Ubiq-F RN-Ubiq-R aaccaaccgtccaacaatcccaac accggatcagcagaggttgatctt 60.1 60.0 CX661133414 z Sequence obtained in the course of this studyy Approximate size in transcripts containing unspliced intronx 5’ portion of transcript sequenced from library using universal primers

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77 Table 3-3. GenBank accession numbers for Fragaria COL genes and other Rosaceae orthologs. Fragaria COL gene Arabidopsis orthologsMalus sp. Prunus persica Prunus dulcis Prunus armeniacaRosa sp. FrCODY675636 DY672035 DY669597 CO380854 AtCO DR996733 CV627730 CO754283 EB132548 EB130204 DR996467 EB141396 EB148115 EB141172 EB129058 CV998001 CV883514 EB141442 CV657475 EB141442 CV657475 DY637206 BU046876 BU046875FrCOL1CX661699 CX661092 CX661512 CX662230 DY674862 CO817106 AtCOL3/4AF052584 (MdCOL1), AF052585 (MdCOL2), and 66 ESTs DY644780 DY643071 DY641101 DY635684 DY635102 DN555580 DN555698 BU042239 (PrpCO) CV049551 CB821542 EC589436 FrCOL2DY669887 DY671835 DY674801 AtCOL5EB157350 and 20 other ESTs BU041471 DY644780 BI977339 BI977724 FrCOL3DY667001 DY673134 AtCOL6/16DN554754 DN553950 BQ641156 FrCOL4DY674124 AtCOL9/10EB144446 EB144014 CN581286 BU044949 DW351298 BQ103878

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78 Figure 3-1. Cladogram of CO-like genes, including all Arabidopsis and full-length Rosaceae COL genes as well as all those from other species Group Ia genes demonstrated to functionally complement Arabidopsis co mutants (underlined). NJ tree was constructed based on the full-length predicted amino acid sequence of each gene. Because the full sequence was not available, FvCOL4 is not included in the analysis, but appears most similar to AtCOL9 and AtCOL10. Strawberry genes are shown in bold. Groups follow the criteria of Griffiths et al., 2003. Lp CO HvCO1 OsHd1 TaHd1-1 PdCO1 PdCO2 FaCO PnCO BnCOa1 AtCO AtCOL1 AtCOL2 FvCOL2 AtCOL5 MdCOL1 MdCOL2 FaCOL1 AtCOL4 AtCOL3 AtCOL9 AtCOL10 AtCOL11 AtCOL12 AtCOL14 AtCOL15 AtCOL13 AtCOL6 AtCOL16 FvCOL3 AtCOL7 AtCOL8 Ia Ic III II

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79 Figure 3-2. Alignment of the predicted amino acid sequence for FraCO with those of AtCO and functionally confirmed dicot homologs, with major conserved domains noted. A tCO MLKQE-------------SNDIGSGENNR-ARPCDTCRSNACTVYCHADSAYLCMSCDAQ 46 BnCOa1 MFKQE-------------SNNIGSEENNTGPRACDTCGSTICTVYCHADSAYLCNSCDAQ 47 PnCO MLKEESCEVLDLDVTIGSSSGSRSGNKQNWARVCDICRSAACSVYCRADLAYLCGGCDAR 60 FaCO MLKEE-------------SNGAAAAN--SWARVCDTCRSAPCTVYCRADSAYLCSGCDAT 45 Consensus M:K:E S.. : : .R CD C S C:VYC:AD AYLC .CDAT A tCO VHSANRVASRHKRVRVCESCERAPAAFLCEADDASLCTACDSEVHSANPLARRHQRVPIL 106 BnCOa1 VHSANRVASRHKRVRVCESCERAPAAFMCEADDVSLCTACDLEVHSANPLARRHQRVPVV 107 PnCO VHGANTVAGRHERVLVCEACESAPATVICKADAASLCAACDSDIHSANPLARRHHRVPIL 120 FaCO IHAANRVASRHERVWVCEACERAPAALLCKADAASLCTACDADIHSANPLARRHQRVPIL 105 Consensus :H.AN VA.RH:RV VCE:CE APA:.:C:AD .SLC:ACD ::HSANPLARRH:RVP:: A tCO PISG--------NSFSSMTTTHHQSEKTMTDPEKRLVVDQEEGEEGDKDAKEVASWLFP157 BnCOa1 PITG--------NSCSSLATANHT---TVTEPEKRVVLVQE-------DAKETASWLFPK 149 PnCO PISGTLYGPPTSNPCRESSMMVGLTGDAAEEDNGFLTQDAEETTMDE-DEDEAASWLLLN 179 FaCO PISG-------------GQIVVGSTPADTTED-GFLSQEGDEEAMDEEDEDEAASWLLLN 151 Consensus PI:G : : : D .E.ASWL: A tCO ---NSD-KNNNNQNNG----------------LLFSDEYLNLVDYNSSMDYKFTGEY--194 BnCOa1 ---NSDNHNNNNQNNE----------------LLFSDDYLDLADYNSSMDYKFTGQYNQP 190 PnCO PNPNPNPNPVKSNNSTNMCKGGNNNNNEMSCAVEAVDAYLDLAEFSSCHNNLFEDKYS-237 FaCO PVKNSNSHNSNNNNNP------NSNNNGFFFGVE-VDEYLDLVEYNSSDQNQFSGTTA—202 Consensus N.: : :.:N. : D YL:L .::.S. : F A tCO SQHQQNCSVPQT--SYGGDRVVPLKLEE-----SRGHQCHNQQNFQFNIKYGSSGTHYND 247 BnCOa1 TQHKQDCTVPEK--NYGGDRVVPLQLEE-----TRGNLHHKQH----NITYGSSGSHYNN 239 PnCO INQQQNYSVPQRNMSYRGDSIVP-NHGKNQFHYTQGLQQHNHH--AIFNCKEWNMRILTR 294 FaCO TNDQHSYGVPHK-ISYGGDSVVPVQYGEGKVTQMQMQQKHNFH--QLGMEYESSKAAYGY 259 Consensus :.::. VP. .Y GD :VP:: : : H: : : : A tCO NGSINHNAYISSMETGVVPESTACVTTASHPRTPKGTVEQQPDPASQMITVTQLSPMDRE 307 BnCOa1 NGSINHNAYNPSMETDFVPEQTAPDKTVSHPKTHKGKIEKLPEPLIQIL-----SPMDRE 294 PnCO D-----MVSISSMDVGVVPESTLSDTSISHSRASKGTIDLFSGPPIQMPPQLQLSQMDRE 349 FaCO DGSISHTVSVSSMDVGVVPDSTMSEMSVCHPRTPKGTIDLFNGLTIQIP--TQLSPMDRE 317 Consensus : : .SM:...VP:.* : .H.:: KG.:: Q: :S MDRE A tCO ARVLRYREKRKTRKFEKTIRYASRKAYAEIRPRVNGRFAKR-EIEAEEQGFNTMLMYN-T 365 BnCOa1 ARVLRYREKKKRRKFEKTIRYASRKAYAERRPRINGRFAKISETEVEDQEYNTMLMYYDT 354 PnCO ARVLRYREKKKTRKFEKTIRYASRKAYAETRPRIKGRFAKRTDVDTEVDQIFYAPLMAES 409 FaCO ARVLRYREKKKTRKFEKTIRYASRKAYAEARPRIKGRFAKRTDIDVEVDQMFSTSLMGET 377 Consensus ARVLRYREK:K RKFEKTIRYASRKAYAE RPR::GRFAKR : :.V : : : A tCO GYGIVPSF---373 BnCOa1 GYGIVPSFYGQK 366 PnCO GYGIVPSF---417 FaCO GYGIVPSY---385 Consensus GYGIVPS: B-Box 1 B-Box 2 M1 M2 M3 M4 CCT

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80 Figure 3-3. Alignment of the predicted amino acid sequence for FrvCOL1 with possible apple and Arabidopsis homologs, with major conserved domains noted. MadCOL1 --------------------------------------------MALKLCDSCKSATGTL 16 MadCOL2 --------------------------------------------MASKLCDSCQSATATL 16 FaCOL1 --------------------------------------------MASKLCDSCKSATATL 16 A tCOL4 MDPTWIDSLTRSCEANSNTNHKRKRERETLKHREKKKKRFRERKMASKLCDSCKSATAAL 60 A tCOL3 ------------------------------------------MASSSRLCDSCKSTAATL 18 Consensus : :LCDSC:S::.:L MadCOL1 FCRADSAFLCVNCDSKIHAANKLASRHARVWLCEVCEQAPAHVTCKADDAALCVTCDRDI 76 MadCOL2 FCRADSAFLCVNCDSKIHAANKLASRHPRVWLCEVCEQAPAHVTCKADDAALCVTCDRDI 76 FaCOL1 FCRADSAFLCINCDTKIHAANKLASRHARVWLCEVCEQAPAHVTCKADDATLCVTCDREI 76 A tCOL4 YCRPDAAFLCLSCDSKVHAANKLASRHARVWMCEVCEQAPAHVTCKADAAALCVTCDRDI 120 A tCOL3 FCRADAAFLCGDCDGKIHTANKLASRHERVWLCEVCEQAPAHVTCKADAAALCVTCDRDI 78 Consensus :CR.D:AFLC .CD K:H:ANKLASRH RVW:CEVCEQAPAHVTCKAD A:LCVTCDR:I MadCOL1 HSANPLSHADERVPVTPFYDSVNSATDSVPAVKSAVNFLNDRYFSDVDGEIEARREEAEA 136 MadCOL2 HSANPLSSRHDRVPVTPFYDSVNSAANSVPVVKSVVNFLDDRYLSDVDGETEVSREEAEA 136 FaCOL1 HSANPLSRRHERVPVAPFYDSLNSGKSDA----AAVNLLDDRYLS--DGE----TTEAEA 126 A tCOL4 HSANPLARRHERVPVTPFYDSVSSDGSVK---HTAVNFLDDCYFSDIDGNGSREEEEEEA 177 A tCOL3 HSANPLSRRHERVPITPFYDAVGPAKSAS----SSVNFVDE------DGG-------DVT 121 Consensus HSANPL: .:RVP::PFYD::.. : FV:::: DG : MadCOL1 ASWLLP-NPKAM------------------ENPDLNSGQ-YLFPEMDPYMDLDYGHVDPK 176 MadCOL2 ASWLLP-NPKAM------------------ENPDLNSGQ-YLFQEMDPYLDLDYGHVDPK 176 FaCOL1 ASWLLP-NPK-----------------------DLNSGQ-YVFSDMDSYLDLDYGTPADP 161 A tCOL4 ASWLLLPNPKTTTTATAGIVAVTSAEEVPGDSPEMNTGQQYLFSDPDPYLDLDYGNVDP236 A tCOL3 ASWLLA-------------------------KEGIEITN--LFS------DLDY----PK 144 Consensus ASWLL :: : :F DLDY MadCOL1 LEDAQEQNSCITDGVVPEQSKNMQPQLVNDHSFEIDFSAASKPFVYGYHHAQCLRQSVSS 236 MadCOL2 LEEAQEQNSCGADGVVPVQSKNMQPLLVNDQSFELDFSAGSKPFVYGYHHARCLSQSVSS 236 FaCOL1 KTEAQEQNSSATDGVVPVQSKSAQP-----QSFEMELPG-SKPFIY-------LSQSVSS 208 A tCOL4 KVESLEQNSSGTDGVVPVENRTVRIPTVNENCFEMDFTGGSKGFTYGGGYN-CISHSVSS 295 A tCOL3 IEVTSEENSSGNDGVVPVQNKLFLN----EDYFNFDLSA-SKISQQGFN---FINQTVST 196 Consensus : E:NS. DGVVP :.: F::::.. SK : ::VS: MadCOL1 SSMDVSIVPDDNAMTDDSNPYNKSMTSAVES-SHPAVQLSSADREARVLRYREKRKNRKF 295 MadCOL2 SSMDISVVPDGNAVT-----------AAVET-SQPAVQLSSVDRVARVLRYREKRKNRKF 284 FaCOL1 SPLDVSIVPDGNMSD----PYPKSISSAVDQLSHPTVQISSADREARVLRYREKRKNRKF 264 A tCOL4 SSMEVGVVPDGGSVADVSYPYGGPATSGADPGTQRAVPLTSAEREARVMRYREKRKNRKF 355 A tCOL3 RTIDVPLVPESGGVT-----------AEMTNTETPAVQLSPAEREARVLRYREKRKNRKF 245 Consensus .::: :VP:.. : :V ::..:R ARV:RYREKRKNRKF MadCOL1 EKTIRYASRKAYAETRPRIKGRFAKRTEVEIEAEPMCR------YGIVPSF 340 MadCOL2 EKTIRYASRKAYAETRPRIKGRFAKRTEVEIEAERMCR------YGVVPSF 329 FaCOL1 EKTIRYASRKAYAETRPRIKGRFAKRTEVEIEAERLCR------YGVVPSF 309 A tCOL4 EKTIRYASRKAYAEMRPRIKGRFAKRTDTNESNDVVGHGGIFSGFGLVPTF 406 A tCOL3 EKTIRYASRKAYAEMRPRIKGRFAKRTDSRENDGGDVG--VYGGFGVVPSF 294 Consensus EKTIRYASRKAYAE RPRIKGRFAKRT: . :G:VP:F B-Box 1B-Box 2 M1 M3 M4 CCT

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81 Figure 3-4. Structures of COL alleles from Fragaria species: F. ananassa ‘Strawberry Festival’ ( Fra ), F. vesca FDP815 ( Frv ), F. nubicola FDP 601 ( Frn ), and F. iinumae FRA377 ( Fri ). Thinner black lines indicate introns, dark grey indicates conserved domains, and crosshatching indicates major indel polymorphism sites. FraCO-C9 362 bp 746 bp 791 bp 688 bp 119 bp 383 bp FrvCOL2 FraCOL1-7C 82 bp 620 bp 322 bp 688 bp 118 bp 383 bp FrnCOL2 FrnCO 703 bp 362 bp FraCO-35 706 bp 791 bp 362 bp FrvCO 704 bp 796 bp 362 bp FriCO 697 bp 799 bp 362 bp 784 bp FrnCOL1 82 bp 620 bp 310 bp FrvCOL1 82 bp 620 bp 310 bp

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82 Figure 3-5. NJ phylogram tree of intron sequence divergence among FrCO alleles.

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83 (A)F. vesca Hawaii-4 CTGTGAA--ACAGC-AATAGCCACAACAGTAACA-ACAACAACAATCCGAACAGT-AAC F. vesca FDP815 CTGTGAAGAACAGC-AATAGCCACAACAGTAACA-ACAACAACAATCCGAACAGT-AAC F. nubicola FDP601 CTGTGAAGAACAGC-AATA----------------ACCACAACAATCCGAACAAT-AAC F. x ananassa ‘Festival’ C9 CTGTGAAGAACAGC-AATA----------------ACCACAACAATCCGAACAAT-AAC F. x ananassa ‘Festival’ 35 ATCTGAATCCTGTGAGACAGCAATAACT-------ACAACAACAATCCGAACAGT-AAC F. bucharica FDP520 ATCTGAATCCTGTCAGGACAGCACCACGCACCAG-------TAACACACAACAATCGAC F. iinumae FRA377 ATGTGAACGGATAACAATTTTCACACAGGAAACAGCTATTG-----ACCCATGAT-TAC ****** * ** F. vesca Hawaii-4 AACAACGGATTCTTCTTCGGAGTGGAGGTTGATGAGTACTTGGACCTTGTGGAGTACAAC F. vesca FDP815 AACAACGGATTCTTCTTCGGAGTGGAGGTTGATGAGTACTTGGACCTTGTGGAGTACAAC F. nubicola FDP601 AACAACGGATTCTTCTTTGGAGTGGAGGTTGATGAGTACTTGGACTTTGTGGAGTACAAC F. x ananassa ‘Festival’ C9 AACAACGGATTCTTCTTTGGAGTGGAGGTTGATGAGTACTTGGACTTTGTGGAGTACAAC F. x ananassa ‘Festival’ 35 AACAACGGATTCTTCTTCGGAGTGGAGGTTGATGAGTACTTGGACCTTGTGGAGTACAAC F. bucharica FDP520 ATCCACCAACGGATCTCTCGGAGTGAGGTTGATGAGTACTTGGACCT-GTGGAGTACAAC F. iinumae FRA377 GGCCAAGCTTGGTACCGAGGCTCCGGATTCCACTAGTAACGGCCGCCAGTGTGCTGGAAT * * * *** ** **(B) Figure 3-6. Comparisons of hypervariable section of coding region in FrCO (A) Alignment of variable region of seven FrCO alleles, (B) NJ phylogram comparing nucleotide sequence within the variable region.

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84 Figure 3-7. Southern blot of genomic DNA from F. ananassa ‘Strawberry Festival’ and F. vesca Hawaii 4, cut with six different enzymes and probed with a portion of FrvCO Lane 1= Xho I, 2= Xba I 3= Hind III 4= EcoR V 5= EcoR I, 6= BamH I 11 2 2 3 344 5 5 66F. ananass a F. vesc a

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85 (A)(B) 1 2 35 47101314 9 681112 564 bpaa bb abab ababababab aa bbbbbbbb 15(C)Figure 3-8. (A) Map of the portion of the FrCO product amplified for use in mapping, showing the locations of distinguishing restriction sites and lengths of the resulting fragments. (B) Gel showing characteristic band patterns after digestion with Hind III for the F. vesca parent (lane 1, genotype aa), the F. nubicola parent (lane 2, genotype bb), twelve members of the F2 (lanes 3-14), and the 564 bp band of the Hind III size standard (lane 15). (C) Partial map of linkage group VI (after Sargent et al. 2005), showing the predicted location of FrCO RN-FaCO F RN-FaCO R Hind III Hind III Hind III Xba I EcoR IFrvCO FrnCO354 bp 354 bp 136 bp 403 bp199 bp 587 bp 154 bp EMFv160BC UFFxa01E03 FAC-012a UDF025FrCOEMFv010 EMFn153 EMFv160AD FAC-005 74.1 72.5 63.7 61.7 60.5 56.0 54.9 54.0 51.6

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86 Table 3-4. Mean inflorescence and runner production of F. ananassa and F. vesca genotypes under LD (16 h) and SD (8 h) photoperiods, at 18/16C day/night temperature. 4 weeks6 weeks8 weeks GenotypeInflorescencesRunnersInflorescencesRunnersInflorescencesRunners Long Day Diamante1.20.03.20.03.80.2 Camarosa0.00.00.00.00.00.0 Earliglow0.02.20.03.00.03.8 Hawaii 4——0.12.80.23.8 Short Day Diamante0.40.00.60.01.00.0 Camarosa0.00.00.30.01.00.0 Earliglow0.02.40.02.80.03.0 Hawaii 4——3.01.83.31.8

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87 Figure 3-9. Northern blot of RNA from ‘Strawberry Festival’, a SD genotype, and ‘Diamante’, a DN genotype, collected every 4 h, showing expression of FraCO under short and long day conditions. Diamante (DN) Diamante (DN) Festival (SD) Festival (SD) Short Day (8 h) Long Day (16 h)

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88 Figure 3-10. RT-PCR assay of expression of FrCO transcript under short (8 h) and long (16 h) days in three genotypes: F. vesca Hawaii-4 (SD, diploid), F. ananassa ‘Camarosa’ (SD, octoploid), and F. ananassa ‘Diamante’ (DN, octoploid). Dawn occurred at 8 a.m. in both chambers, and dusk was at 4 p.m. in the short day chamber and at midnight in the long day chamber.Short Day (8 h L) Long Day (16 h L)11p 3am 7am 11am 3pm 7pm Camarosa (SD) Diamante (DN) Hawaii-4 (SD) Camarosa (SD) Diamante (DN) Hawaii-4 (SD)

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89 Figure 3-11. Expression of 35 and C9 alleles of FraCO at dawn under SD, using RT-PCR at 33 and 40 cycles, in ‘Strawberry Festival’ (F) and ‘Diamante’ (D). FraCO-C9 FraCO-35F DF D 33 cycles 40 cycles

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90 Table 3-5. Sources of plants used in this chapter. GenotypeSpeciesSourceLocation Camarosa F. ananassa Garden Gate NurseryGainesville, FL Earliglow F. ananassa Edible LandscapingAfton, VA Diamante F. ananassa Edible LandscapingAfton, VA Strawberry Festival F. ananassa GCRECWimauma, FL Hawaii-4 F. vesca University of N.H.Durham, NH

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91 Figure 3-12. RT-PCR assay of FrCOL2 expression in F. vesca Hawaii-4, showing variation in splicing efficiency at several points under short days. Hawaii-4 (SD)11pm 3am 7am 11am 3pm 7pmShort Day (8 h L)

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92 Figure 3-13. Ubiquitin controls after equalization of RT-PCR samples using RN-Ubiq1 primer set to standardize template amounts. Camarosa (SD) Diamante (DN) Hawaii-4 (SD) Short Day (8 h L)Long Day (16 h L) 11pm 3am 7am 11am 3pm 7pm 11pm 3am 7am 11am 3pm 7pm

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93 CHAPTER 4 IDENTIFICATION AND CHARACTERIZATION OF FLOWERING-RELATED GENES IN Fragaria AND OTHER ROSACEAE Introduction Although CONSTANS plays a central role in the integration of photoperiod and the circadian clock into the regulation of flowering, CO itself does not directly control the reproductive transition. Instead, CO along with other inputs, regulates a network of proteins which through a cascade of interactions eventually trigger the activation or repression of a suite of meristem identity genes that ultimately govern the developmental fate of the meristem. In Arabidopsis, the two most important regulators of flowering directly downstream from CO are FLOWERING LOCUS T ( FT ) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( SOC1 ) (Samach, 2000). These two proteins act in parallel to promote flowering, and are affected by different environtmental inputs. A third protein, TERMINAL FLOWER 1 ( TFL1) is closely related to FT but has an opposing role. Rather than promoting a transition to flowering in the meristem, TFL1 delays it, causing growth to remain vegetative (Kobayashi et al., 1999). FT and TFL seem to have fairly direct impacts on the meristem, but SOC1 acts primarily through the activation of protein called LEAFY ( LFY ) to promote flowering. FT appears to be under greater photoperiodic control than SOC1 (Moon et al., 2005) Although promoted by CO SOC1 is also under regulation by the other pathways regulating flowering: vernalization, autonomous, and gibberellin, and serves as the point at which these regulatory networks integrate into the general flowering pathway (Izawa et al., 2003). In addition to these components, a number of other genes affect flowering as well. These include components of the circadian clock (Turner et al., 2005) and photoreceptors (Izawa et al.,

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94 2003), which affect CO transcript and protein levels, as well as components of the other floral pathways. Mutations in these genes cause significant changes in flowering time and sensitivity to environmental inputs in Arabidopsis, as well as in crop plants such as rice (Izawa et al., 2003). Although the genetic basis for variation in flowering habit in strawberry is currently unknown, it would seem quite possible that differences in the expression of this small suite of genes lie at the core of it. By identifying and characterizing the orthologous genes in Fragaria it may be possible to pinpoint the location of the mutation or mutations involved in the shift from short day to everbearing or day-neutral flowering. This could in turn be the basis for molecular markers to assist in breeding, a target for genetic engineering, or even shape cultural practices by clarifying the interaction of the different physiological mechanisms. Such knowledge may have impacts even outside of strawberry, particularly for the other important crops within the Rosaceae. To that end, efforts were made to identify the genes comprising this network in Fragaria and the expression of some of these genes was examined in strawberry plants under long and short day conditions. Results Identification of Rosaceae Flowering Gene Homologs Screening of Fragaria EST libraries developed in this laboratory (Folta et al., 2005, as well as others as outlined in Chapter 3) revealed a number of sequences related to transcripts known to be involved in flowering in Arabidopsis rice, or apple (Tables 4-1 & 4-2). In addition to Fragaria transcripts, ESTs from other Rosaceae species were also searched for MADS-box and FT/TFL1-like genes, in order to gain insight into the nature of these genes and gene families within the larger taxon, and to help fill in gaps where no strawberry genes are known but may exist.

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95 MADS-Box genes Six strawberry MADS-Box genes were identified in ESTs, adding to the three already documented in GenBank. Most had fairly clear correspondence with one of the fifteen documented MADS-Box genes in apple, although not all had clear homologs in Arabidopsis. These included genes related to the Arabidopsis transcription factors SOC1 SEPALLATA3, PISTILLATA, SHORT VEGETATIVE PHASE, SHATTERPROOF1/2, and AGAMOUS-LIKE14 as well as one transcript not closely related to any Arabidopsis gene but somewhat similar to the apple MdMADS11 These were designated, in order, FraSOC1, FrvSEP3, FrvPI, FrvSVP, FrvMADS1, FrvMADS2, and FrvMADS3 These genes are listed in Table 1, and their implied relationship to the other known Rosaceae MADS-Box genes and a selection of Arabidopsis MADS-Box genes are shown in Figure 1. FraSOC1 was first identified as a partial transcript (GenBank Accession CO817776) resembling a MADS box transcription factor among a small EST library developed from whole plant tissues of F. ananassa ‘Strawberry Festival’ (Folta et al., 2005). Because the glycerol stock of this clone was available, we were able to sequence the gene from the other end, obtaining the entire sequence. The 642 bp coding region encoded a predicted MADS Box transcription factor, with high similarity to the Arabidopsis SOC1 with 63% identity at the amino acid level. The closest overall match annotated in the public databases was VvMADS8 again from grapevine ( Vitis vinifera L.), to which it shared 68% identity at the amino acid level. Other than this strawberry gene and the previously identified relative in apple (Valazideh et al., 2006), no apparent SOC1 homologs were found among Rosaceae ESTs. A second Fragaria gene, FrvMADS3 (GenBank Accession DY675364), also grouped within the SOC clade, but more closely resembled AGL14 than SOC1 .

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96 FT/TFL1 genes An EST (DY674124) bearing considerable similarity to the floral repressors TFL1 and ATC was also identified among sequences derived from cold-stressed seedlings of the F. vesca selection Hawaii-4. This transcript was initially passed over because BLAST-X searches showed only partial similarity to known genes. Upon closer inspection, it became clear that the transcript retained 147 bp of intron sequence at a site near the middle of the gene, which resulted in a frameshift, causing multiple stop codons and corrupting more than half of the predicted protein. This likely represents only a partial failure to complete splicing, as the apple (DQ535888) and Arabidopsis (NM_120465) genes each contain three introns. With the exon sites identified by comparison to these likely homologs, the spliced transcript yielded a coding region of 666 bp and encoded a protein with 83% identity to ARABIDOPSIS THALIANA CENTRORADIALIS ( ATC ), and a lower 63% identity to the Arabidopsis TFL1 (Figure 4-2), although the proportion of positive matches, 91% and 85%, respectively, were much closer. The most similar annotated transcript was from Populus a “Flowering Locus T-like protein” (BAD22601), with which the protein shared 85% identity. At the nucleotide level, the most similar annotated sequence was again from Poplar, PnTFL1 (AB181184), with an e-value of e-60, and once again this transcript was significantly more similar than the nearest Rosaceae gene, MdTFL1 1 (AB052994), with an e-value of 9e-28. Efforts to amplify a strawberry ortholog of FT from genomic or cDNA using degenerate primers based on apple and Arabidopsis sequence were unsuccessful, as was colony hybridization with Arabidopsis FT as a probe, and no likely candidates were identified in any of the EST libraries. No Fragaria sequences were identified with significant similarity to the other members of the Arabidopsis family: TSF, MFT ( MOTHER OF FT ), and BFT ( BROTHER OF FT ), however apple ESTs resembling MFT and BFT were identified (GenBank Accessions

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97 EB134193 and EB138045, respectively), as well as a partial transcript of a MFTlike gene from almond (GenBank accession BU574411). Relationships of apple and strawberry amino acid sequences with members of the FT/TFL1 family in Arabidopsis are shown in Figure 4-2. Other genes thought to be involved in flowering Also identified were a number of elements of the circadian clock, including apparent orthologs of several response and pseudo-response regulators, TOC1, FKF1 ZTL as well as several genes with roles in photomorphogenesis, including genes similar to HY5 and hemeoxygenase (Table 1). Attempts to use degenerate primers based on consensus sequence from apple, pear, and Arabidopsis LEAFY genes to amplify the strawberry ortholog from either cDNA library or genomic DNA were not successful. However, a fosmid from F. vesca containing the genomic sequence of LEAFY was obtained from Dr. Thomas Davis of the University of New Hampshire. FrSOC1 Expression Surprisingly, RT-PCR assays showed FrSOC1 expression did not parallel flowering as expected. Rather, in ‘Camarosa’, FrSOC1 generally followed FrCO expression under short days, but with a lag of several hours, with a peak near the end of the day and tapering off into the night. Under long days, however, the expression levels in all but ‘Diamante’ were extremely high, and remained so through out the course of the day (Figure 4-3). In ‘Diamante’, the dayneutral genotype, however, FrSOC1 expression followed similar patterns under both conditions. FrTFL1 and FrLFY Expression Because of the unexpected results seen with FrSOC1, RT-PCR assays of FrLFY expression were also conducted on the short day octoploid cultivar, Camarosa. Although expression levels were low, an increase to 36 cycles revealed expression of FrLFY (Figure 4-4) and FrTFL1 (Figure 4-5) to be the opposite of that anticipated by the Arabidopsis model, just as

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98 with FrSOC1 Under short days, when ‘Camarosa’ was initiating flowers, neither was detectable in leaf tissues. Under long days, when growth was primarily vegetative, however, both were expressed at all time points, though levels were low. Discussion Rosaceae MADS-Box Genes MADS-box genes play critical roles in the a number of plants processes, especially in the development of fruits and flowers (Becker and Theissen, 2003), and as such are important targets of study in crop plants. The Arabidopsis MADS-box gene family comprises roughly a hundred members, though only one group, the so-called MIKC class which contains 39 Arabidopsis genes, has been functionally characterized to a significant extent (Becker and Theissen, 2003). Becker and Theissen (2003) break this class into fourteen groups, one of which, TM8, does not exist in Arabidopsis. The eight Fragaria MADS-box transcription factors identified in this study along with those identified by previous researchers represent seven of these groups. An eighth group, AP1-like MADS-box factors, is represented in several other species within the family, including peach ( PrpMADS6 ), loquat ( EjAP1 ), almond ( PrdMADS2 ), and several genes in apple ( MdAP1, MdMADS5, MdMADS2, MdMADS12, and MdMADS12a ), but none are yet known in strawberry. The six groups not yet attested in any of the Rosaceae are the AGL12 AGL15 TM8 TT16 ANR1 and FLC groups. Of these, FLC is the best characterized, playing a critical role in the regulation of flowering by non-photoperiodic factors, especially vernalization (Boss et al., 2004). It is worth noting that FLC orthologs have been identified only within the Brassicaceae, and are not functional within all ecotypes of Arabidopsis, nor have AGL15 genes (Becker and Theissen, 2003). With the exception of the ANR1 group, none of these groups were attested in the legume species Medicago, soybean, and lotus (Hecht et al., 2005). The absence of AGL12, AGL15, and

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99 ANR1type genes may be explained by the absence of root-derived Rosaceae ESTs, as these genes appear to be primarily active in root development (Becker and Theissen, 2003). The remaining groups are mostly poorly characterized in Arabidopsis. Among the most numerous Rosaceae MADS-box genes are those of the SEPALLATA group. The SEP group in Arabidopsis consists of four members that interact with other MADSbox proteins to shape organ identity in the four whorls of flower development (Ditta et al., 2004). Two such genes were identified in strawberry, FraMADS-RIN (Vrebalov and Giovannoni, 2002), with greatest similarity to AtSEP1 and AtSEP2 and a F. vesca EST we designated FrvSEP3 AtSEP1 and AtSEP2 are very similar to each other, and likely represent recent duplications, and this may account for the seven apple genes in this group, although in the case of apple, these duplications may also be the result of polyploidization in the evolution of this species’. The bulk of the Rosaceae SEP genes most closely resemble the SEP1/SEP2 genes of Arabidopsis, including FraMADS-RIN peach and almond genes, and all but one of the apple genes in the group. FrvSEP3 and the pear gene PbMADS2 (GenBank Accession AB265800) most closely resemble SEP3 whereas MdMADS4 does not bear clear relation to a specific Arabidopsis member, though it most closely resembles SEP4 In Arabidopsis, the SEPALLATA genes appear to be largely redundant in function, and plants lacking all three produce flowers composed solely of sepals (Pelaz et al., 2000). Sung et al. (2000) investigated the expression of two such genes from apple, MdMADS3 and MdMADS4 and found very different expression patterns, with the first expressed only in the floral primordium, whereas the second was expressed throughout floral and early fruit development, particularly in vascular bundles. Yao et al. (1999) found seven MADS box genes were expressed in distinct patterns during flower and fruit development. It seems likely that Fragaria genes within this family also play roles in reproductive development,

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100 although the developmental differences between Arabidopsis, apple, and strawberry fruit require some caution in predicting function based on these other species. One new strawberry MADS-box gene, designated FrvMADS3 shares some similarities with the SEP subfamily, but appears most closely related to the AGL6 group, which it shares with the apple gene MdMADS11 The function of these genes is poorly understood. In Arabidopsis, their expression seems to be confined to floral organs (Becker and Theissen, 2003) There is evidence that OMADS1 an AGL6-type gene from orchid, interacts with OMADS3 a factor involved in flower formation, and promotes flowering when expressed ectopically through the upregulation of SOC1 and FT in Arabidopsis (Hsu et al., 2003). Similar results were achieved by Tian et al. (2005) in their study of another AGL6-like gene, DlMADS18 from bamboo ( Dendrocalamus latiflorus Munro). The AP3 and PISTILLATA class genes are class B organ identity genes, and combine with class A and C genes to specify petals and stamens, respectively (Krizek and Meyerowitz, 1996). Both have been previously identified in apple (Yao et al., 2001) and rose (Kitahara et al., 2001). The three apple mutants—‘Rae Ime’, ‘Spencer Seedless’, and ‘Wellington Bloomless’—which produce apetalous flowers and parthenocarpic fruit, were found by Yao et al. (2001) to contain retrotransposon insertions in introns of MdPI, abolishing expression and suggesting a critical role for the gene in both fruit and flower development. The strawberry PI gene identified here might thus be an interesting target for RNAior antisense-based suppression of expression. Parthenocarpy might be a means to limit malformation of fruit, which can result from incomplete pollination, and might also increase fruit size. The gene designated FrvSVP most closely resembles the SVP group of MADS-Box genes. This group appears to play a role in the control flowering in Arabidopsis, with both SVP

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101 (Hartmann et al., 2000) and AGL24 (Yu et al., 2002) acting as repressors of flowering. SVPlike genes have been identified in model legumes (Hecht et al., 2005) and in apple and peach. A peach gene, closely resembling the strawberry FrvSVP (D. Bielenberg, personal communication), has been identified as among the genes deleted in the evergrowing mutation in peach (Bielenberg et al. 2004). The apple gene, MdJOINTLESS (Heo et al., 2006), differs significantly from those of strawberry and peach, more closely resembling the tomato gene of the same name. The final major group are the SOC1 like genes. The role in flowering of SOC1 the best known of these genes, has been described earlier in this work. Related genes in apple (Mahna et al., 2006), rice (Lee et al., 2004), eucalyptus ( Eucalyptus grandis Hill ex. Maiden) (Watson and Brill, 2004) and aspen (Cseke et al., 2003) have been identified and demonstrate evidence of similar functions. VvMADS8 from grapevine, to which FraSOC1 is most similar, has recently been demonstrated to hasten flowering in transgenic Arabidopsis, supporting the idea that it may be that species equivalent of SOC1 (Sreekantan and Thomas, 2006). In some cases, such as eucalyptus (Watson and Brill, 2004) and pea (Hecht et al., 2005), there are multiple family members that appear to fill the role of SOC1 The other members of the SOC1 subfamily in Arabidopsis are not well-characterized, but at least two, AGL14 and AGL19 are expressed solely in roots (Rounsley et al., 1995; Becker and Theissen, 2003). FrvMADS2 identified among F. vesca seedling ESTs, is roughly equal in similarity to both of these genes, and represents the first close relative identified in the Rosaceae. The FT/TFL1 Gene Family in Rosaceae The FT/TFL1 gene family, as defined in Arabidopsis, Antirrhinum and tobacco, is comprised of two clades, the FTlike genes and the TFL1 -like genes, as well as MOTHER OF FT ( MFT ) which is more distantly related to both (Kobayashi et al., 1999). The TFL1 clade is further

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102 divided into the TFL1 -like and CENlike groups. The sole TFL1 -like gene identified in strawberry, FrTFL1 most closely resembles the gene Arabidopsis Thaliana Centroradialis ( ATC ), a member of the CEN group. Although both are very similar genes in both structure and apparent function, ATC and TFL1 differ primarily in the timing of their expression, as ATC is confined to young seedlings while TFL1 is present in mature Arabidopsis plants (Mimida et al., 2001), and as a result, only TFL1 plays a role in governing the reproductive transition. Despite the similarity to ATC a decision was made to designate this gene FrTFL1 following the convention of Carmona et al. (2007). Although FT ATC, and TFL1 are members of the same gene family (Kobayashi et al., 1999; Mimida et al., 2001), the annotation of the Populus ortholog as “FT-like” is probably something of a mis-annotation, since the e-value when compared to Arabidopsis FT was only 1e-50, and 5e-48 when compared to TWIN SISTER OF FT ( TSF ), and all BLAST-X matches higher than the Arabidopsis TFL1 except the Populus gene were annotated as either “TFL1-like” or “CEN-like” ( CEN being the TFL1 homolog in Antirrhinum (Pnueli et al., 1998)) TFL1 orthologs are well-documented in the Rosaceae, though the work is confined to the Maloideae. Consistent with the polyploid origins of this subfamily, Esumi et al. (2005) identified two closely related TFL1 genes in each of six maloid fruit tree species. These genes were expressed in the flower buds of all six species, and elsewhere in few of the species, including in the peduncles of pears and the stamens of apples. Most studies of TFL1 in Rosaceae have focused on its possible role in maintaining juvenility in fruit trees, an important factor given the long delay before fruiting seen in many species. Kotoda and Wada (2005) found a clear relationship between the juvenile state and expression of MdTFL1 and, by using an antisense

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103 construct with this gene, were able to significantly shorten the juvenile period in transgenic ‘Orin’ apples (Kotoda et al., 2006). Juvenility is not a major concern in strawberries, but AtTFL1 ’s role as a determinant of meristem identity may suggest a possible function for FrTFL1 in determining switch between vegetative growth and terminal inflorescence in strawberry. In Arabidopsis, tfl1 mutants flower quickly and terminate inflorescence growth immediately in a single terminal flower whereas 35S:: TFL1 constituitive overexpression lines had extremely long vegetative phases and many axillary buds that would normally have terminated in flowers instead formed long shoots lacking cauline leaves (Radcliffe et al., 1998). Such a transition from terminal inflorescence to elongated axillary shoot seems reminiscent of the switch from flowering to runnering observed in strawberry, and it would seem possible that homologs of TFL1 might be involved in the process. It is worth noting that the subgroup involved in inflorescence develoment, either CEN-like or TFL1-like varies among species, with the CEN genes filling these roles in Antirrhinum tomato, and possibly tobacco, prompting Mimida et al. (2001) to speculate that while the two subgroups diverged before the origination of the angiosperms, it was not until later that one or the other group was “recruited” to participate in inflorescence development. This difference exists solely through the expression of the genes, not through their function, as ATC has been shown to complement tfl1 loss-of-function mutants and vice versa (Mimida et al., 2001). Although the original transcript was obtained from seedling tissues, the RT-PCR results show it to be expressed to a limited extent in mature leaf tisses as well, so it is possible that in Fragaria it is the CEN -like group which is involved in inflorescence development. However, preliminary work reported by Esumi et al. (2006) suggests that in pear and quince trees, expression of TFL1 in the meristem correlates with the development of simple versus complex inflorescences, and a more

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104 TFL1 -like gene, rather than the CENlike one described in this study, may be controlling such development in Fragaria Despite many efforts, no FT sequences were obtained from strawberry species. An apple homolog of FT has previously been characterized (Kotoda and Wada, 2005), and several orthologs of FT other related genes have been identified among apple, peach, and almond ESTs (although the peach FT mapped by Silva et al., 2005 more closely resembles SEP1 ), so it seems unlikely that the family would be completely unrepresented in strawberry. More likely the issue is simply one of very low levels of transcript and a collection of EST data that is still relatively small compared to other species (as of March 3, 2007. there are 19,091 Fragaria ESTs, compared to 91,745 for Prunus and 260,927 for Malus ). Only two FT transcripts could be located among apple ESTs, so it is not surprising that it has not appeared in less than a tenth as many strawberry sequences. The FT subfamily in apple is also represented by orthologs of MFT and BFT Neither gene is well-characterized in Arabidopsis, but MFT has been demonstrated to accelerate flowering, and may serve a role similar to that of FT, albeit at a lower level, whereas overexpression of BFT has no apparent effect on flowering (Yoo et al., 2004). It is interesting that these two genes, apparently of relatively minor importance in Arabidopsis are preserved in apple (and MFT, at least, in almond), indicating that they are both ancient and persistent. Yoo et al. (2004) suggest that differences in MFT expression between Arabidopsis ecotypes may imply an adaptive role, despite a largely redundant function. The fact that it is retained in a relatively distantly related family may support this idea.

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105 Other Flowering Genes A number of components of the circadian clock in strawberry were also identified. Defects in circadian function have been shown to alter flowering time in a number of species (Schaffer et al., 1998; Yamamoto et al., 2003; Zhao et al., 2005), and the day-neutral trait in barley has been shown to be the result of a mutation in Ppd-H1 a pseudo-response regulator that is part of the circadian clock (Turner et al., 2005) Several of these response and pseudo-response regulators were identified, including one, a relative of ARR9 that was mapped in the FVxFN diploid reference population (Sargent et al., 2005). However, the location, at the top of linkage group II, did not correspond to either of the two QTLs for flowering time known in that population, (Sargent et al., 2006). A pseudo-response regulator similar to the Arabidopsis APRR7 ortholog of Ppd-H1 was also identified among F. vesca ESTs. Very few genes related to photoreceptors were identified among the ESTs or by conserved primers. Although hemeoxygenase is involved in the phytochrome chromophore biosynthesis (Muramoto et al., 1999), no phytochrome or cryptochrome genes were identified, nor were other flowering-related photoreceptors with the exception of FKF1 Thus, it remains somewhat difficult to understand the dynamics that underlie the unusual reaction of strawberry plants to red and far-red light demonstrated by Vince-Prue and Guttridge (1973) and Kadman-Zahavi and Ephrat (1974) and outlined in the previous chapter. Although careful spectral studies, such as these, can determine the gross physiology of these interactions, sequence data would allow more targeted studies of the mechanics at work. FrSOC1 FrLFY and FrTFL1 Expression The results of the RT-PCR studies are perhaps the most surprising of this project, and would seem difficult to adequately explain without additional information. It was anticipated that expression of FrSOC1 and FrLFY would parallel the occurence of flowering, as their orthologs

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106 in Arabidopsis (Samach, 2000) do so. In both cases, expression of both genes are upregulated under photoperiods conducive to flowering: long days in Arabidopsis and short days in rice. There is evidence that SOC1 acts at least in part by upregulating LFY (Moon et al., 2005), so it is not surprising that the two genes show similar expression patterns, though no FrLFY transcript is in evidence under short days, despite significant FrSOC1 expression. The rice ortholog of LFY RFL, does not seem to directly promote flowering itself, and instead seems to be down-regulated in flowering plants. Overexpressing this gene in rice inhibits flowering, unlike AtLFY which promotes the conversion of floral meristems into flowers (Kyozuka et al., 1998). Because no Fragaria equivalent to FT could be identified, we are only able to see part of the floral induction network model described in Arabidopsis. It is possible that the primary stimulus of flowering comes through that part of the pathway, as FT has been shown to be able to trigger flowering through genes other than SOC1 and LFY (Yoo et al., 2005). What is perhaps more puzzling is the fact that upregulation of the other two floral integrators, SOC1 and LFY appear not to trigger flowering, but might correlate better with runnering. Although the FrTFL1 data is also the reverse of what was anticipated, the extremely low amounts seen may not be meaningful levels of expression. The rarity of this transcripts suggests that it may localized elsewhere in the plant, and that this may indeed be a homolog of CEN rather than TFL1 and thus may be expressed primarily during early hypocotyl development (Mimida et al., 2001). Another reason these results are unexpected is the relationship they bear to FrCO expression. CO and its orthologs have been shown to play either of two roles. In Arabidopsis and other long day plants, it serves as a promoter of flowering by upregulating FT, SOC1 and, indirectly, LFY (Samach, 2000), but in rice and many other short day plants, however, it

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107 functions to suppress expression of these same genes (Kojima et al., 2002). In the previous chapter, it appeared that because high levels of FrCO were only present during short days, when strawberry was flowering, that it must function as a promoter of flowering. The expression of FrSOC1 under short days, which appears to roughly track expression of FrCO would seem consistent with that analysis, if not for the fact that expression is considerably higher under the non-inductive long day conditions. Nor does LFY a component of the Arabidopsis network downstream from SOC1 appear to be activated at all by high levels of FrCO expression. This would suggest one of three possibilities: either control of flowering is dependent on other integrators, perhaps FT that the floral stimulus conveyed by FrSOC1 and FrLFY is being gated further downstream along the pathway, or that the effects of FrSOC1 and FrLFY differ from those of their Arabidopsis counterparts. In apple, another Rosaceae species, two LFY homologs have been identified: AFL1 and AFL2 These two genes have been shown to promote flowering when overexpressed in transgenic Arabidopsis, as does the native LFY (Wada et al., 2002). However, their overexpression in transgenic apple plants does not accelerate flowering (Kotoda et al., 2006), suggesting that while they are expressed in floral buds, perhaps LFY -type genes do not necessarily function as floral integrators in that species. Because LFY plays another role as a regulator of floral organ differentiation (Simon et al., 1996), it is possible that the gene has retained only this function in the Rosaceae, while losing its role as an integrator of floral signalling, which might explain why the gene has been preserved and expressed. No such other role is known for SOC1 although complex interactions are not unknown among MADS-box genes.

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108 Apple, however, appears to be a day-neutral species (Carew and Battey, 2005), at least in regards to flowering. Although photoperiod seems to play a role in controlling dormancy in Prunus species (Besford et al., 1996), there appears to be little evidence of photoperiod sensitivity in regards to flowering in Prunus species, although there is some suggestion that flowering in at least peach and sand cherry ( P. besseyi L.H. Bailey) is inhibited by short photoperiods (Aerov, 1963). Clear short-day photoperiodic initiation of flowering in the Rosaceae would seem to be limited to the Rosoideae subfamily, as seen in strawberry, biennialfruiting raspberry (Williams, 1959, 1960), and the cinquefoil species Potentilla glandulosa and P. fruticosa (Ahmadi et al., 1991), although some members, such as roses, appear to be normally day-neutral (Zieslin and Mor, 1990). This raises the possibility that photoperiodic flowering in the Rosaceae may have been lost during the course of evolution, and re-emerged in one branch of the family through a novel mechanism. This would explain the striking differences with previously documented systems in other species. This might also explain unsual spectral sensitivities as well (Vince-Prue and Guttridge, 1973). The differences in the photoperiod pathway between Arabidopsis and rice demonstrate how a similar set of parts can be re-tooled by evolutionary forces to produce a very different result, and it seems likely that any novel mechanism would make use of many of the existing elements of the pathway, though perhaps in somewhat modified roles. As with the FrCO portion of this study, one aspect in which our understanding is lacking is at the protein level. High transcript levels do not necessarily mean high levels of protein, nor do low transcript levels necessarily mean low levels of protein, as many factors have been shown to affect the stability and persistence of proteins (Valverde et al., 2004). It may be that while expression of FrSOC1 and FrLFY transcripts are high, the resulting proteins are being rapidly

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109 degraded or otherwise prevented from functioning. However, it must be noted that no such posttranslational regulatory mechanisms involving these particular genes appear to have been previously reported in other species. Methods and Materials Identification of Rosaceae Flowering Genes Predicted amino acid sequences of Arabidopsis genes were queried against GenBank EST databases via the BLAST-X algorithm (Altschul et al. 1997), with the output limited to the Rosaceae. These genes included FT TSF, MFT, BFT, TFL1, TFL2, ATC, PFT1, FKF1, ZTL, TOC1, CCA, LHY, PHYA, PHYB, PHYC, PHYD, PHYE, CRY1, CRY2, LFY, SOC1, the MADSBox domains of AP1 AG, and SEP3, and the CCT domains of CO and TOC1 Additionally, the twenty published apple MADS-box gene nucleotide sequences ( MdMADS1-14, MdPI, MdJOINTLESS, MdSOC1, MdAG, MdAP1 and MdTM6 ) were also queried against Rosaceae EST sequences, using the BLAST-N protocol (Altschul et al., 1997), as were the apple genes AFL1, AFL2, MdTFL1-1, MdTFL1-2, and MdFT Some Fragaria flowering genes, including heme-oxygenase, FraHY5 and FraSOC1 were noted by visual inspection of BLAST-X results lists prepared during analysis of a small F ananassa library (Folta et al., 2005). Phylogenetic Tree Construction Trees were constructed based on full-length amino acid sequence using the “Neighbor Joining” (NJ) method of Saitou and Nei (1987), through the CLUSTAL-W alignment program (online at http://www.ebi.ac.uk/clustalw/) (Higgins et al., 1994). The resulting DND data file was saved and exported to TreeEdit 1.0a10 Carbon for MacOS X, which was used to draw the trees. RNA Extraction and RT-PCR Experiments RNA was extracted from mature leaf tissue using a modification of Chang et al. (1993), as described in Folta et al. (2005) and in Chapter 3. RT-PCR experiments were conducted as

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110 described for the second FrCO/FrCOL expression experiment in Chapter 3, with the exception of FrLFY for which 35 cycles of PCR were used.

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111 Figure 4-1. NJ tree showing similarity between full-length amino acid sequences of Rosaceae and Arabidopsis MADS-Box genes. Md is Malus x domestica Prp is Prunus persica Prd is Prunus dulcis Pyb is Pyrus x bretschneiri Rr is Rosa rugosa Rh is Rosa hybrida Ej is Eriobotrya japonica and Frv and Fra are F. vesca and F. ananassa respectively.

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112 Table 4-1. Identified Fragaria genes related to flowering and photoperiodic response, with the closest match at the amino acid level among Arabidopsis and all annotated transcripts. Strawberry Gene Arabidopsis Match E Value Closest Match E Value FrvTFLATC6e-73Flowering Locus T-like protein ( Populus nigra ) 6e-83 FraSOC1SOC11e-45VvMADS8 ( Vitis vinifera )1e-54 FrvLFYLFY9e-103LFY-2 ( Cydonia oblonga )2e-123 FrvFKF1FKF17e-42FKF1 ( Arabidopsis thaliana )7e-42 FrvPIPI7e-55MASAKO BP ( Rosa rugosa )2e-87 FrvSVPSVP9e-38MADS1 ( Populus tomentosa )5e-43 FrvAGL14AGL142e-52VvMADS8 ( Vitis vinifera )1e-53 FrvSEP3SEP32e-90PbMADS2 ( Pyrus x bretschneideri ) 5e-113 FrvMADS11AGL65e-72VvMADS3 ( Vitis vinifera )2e-94 FrvMADS14AGL1/SHP15e-80MASAKO D1 ( Rosa rugosa )1e-115 FraHY5HY54e-47HY5 ( Arabidopsis thaliana )4e-47 FrvTOC1TOC15e-70TOC1 ( Arabidopsis thaliana )5e-70 FraRR9/FrvRR9ARR93e-31Response regulator receiver ( Medicago trunculata ) 93-35 FrvPRR5APRR51e-56APRR5 ( Arabidopsis thaliana )1e-56 FrvPRR7APRR78e-81APRR7 ( Arabidopsis thaliana )8e-81 FrvZTLZTL1e-95Spercat/scavenger receptor; cylinlike F-box; galactose oxidase, central ( Medicago trunculata ) 4e-104

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113 Figure 4-2. NJ phylogeny tree showing relationships between Arabidopsis and Rosaceae members of the FT/TFL1 gene family, based on full predicted protein sequence.

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114 Table 4-2. RT-PCR primers for strawberry flowering genes and controls, sequence source, TM, and approximate size in cDNA (as calculated from sequence).Gene PrimersSequence TM (C) ESTbp Flowering Genes FrSOC1 RN-FrSOC1-F RN-FrSOC1-R cgcaagctcgagcatgcagg aagctagtgcttcgatctcc 61.8 54.2CO817776481 FrLFY FvLFYintron2-F FvLFY-R agcgtggtgaaaaatgtcccacc tcagtagggcagctgatgagc 60.3 59.1 — z409 FrTFL1 RN-TFL1-F2 RN-TFL1-R agcttgcagcttcagaaagagc tggcacgtacaagtgcttccaa 58.1 59.4DY674124(246)y403 Control Genes FrActin1 RN-Actin-F RN-Actin-R tggctgtgcacgatgattgc taacttcccaccagatatcc 58.8 50.9 DV439971430 FrUbiq1 RN-Ubiq-F RN-Ubiq-R aaccaaccgtccaacaatcccaac accggatcagcagaggttgatctt 60.1 60.0 CX661133414 z Sequence obtained from fosmid sequenced by Tom Davis, U.N.H.y Predicted size of transcripts when fully spliced

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115 Figure 4-3. RT-PCR assay of expression of FrSOC1 under short (8 h) and long (16 h) photoperiods in ‘Camarosa’ (SD, octoploid) and ‘Diamante’ (DN, octoploid).11pm 3am 7am 11am 3pm 7pmCamarosa (SD) Diamante (DN) Camarosa (SD) Diamante (DN) Short Day (8 h L) Long Day (16 h L) 11pm 3am 7am 11am 3pm 7pm

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116 Figure 4-4. RT-PCR assay of expression of FrLFY under short (8 h) and long (16 h) photoperiods in Hawaii-4 (SD, diploid) and ‘Camarosa’ (SD, octoploid).11p 3am 7am 11am 3pm 7pmCamarosa (SD) Hawaii-4 (SD) Camarosa (SD) Hawaii-4 (SD) Short Day (8 h L) Long Day (16 h L) 11p 3am 7am 11am 3pm 7pm

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117 Figure 4-5. RT-PCR assay of expression of FrTFL1 under short (8 h) and long (16 h) photperiods in Hawaii-4 (SD, diploid). 11p 3am 7am 11am 3pm 7pm Hawaii-4 (SD) Hawaii-4 (SD) Short Day (8 h L) Long Day (16 h L) 11p 3am 7am 11am 3pm 7pm

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118 CHAPTER 5 Fragaria ALLELES OF POLYGALACTURONASE INHIBITOR PROTEIN GENES Introduction At any given moment, a plant’s environment is full of potentially pathogenic organisms. The fact that the vast majority of these fail to result in disease is a testament to the effectiveness of plants’ many-faceted defensive mechanisms. Yet these dangers represent diverse and rapidly evolving threats to a plant survival. Furthermore, pest species are undergoing continual selection for their ability to best exploit the broad range of hosts available in the environment (Thrall and Burdon, 2003). Similarly, plants in the wild are undergoing natural selection for their ability to recognize and counter this changing threat (Stotz et al., 2000). In cultivated plants, this need for selection falls instead on the plant breeder and the farmer. This interaction is reflected in the “gene-for-gene” theory of resistance, a dominant paradigm in plant pathology for nearly half a century. This theory, first developed by Flor (1956), through his work on rust resistance in flax, proposes that for each resistance gene there is a matching avirulence gene in the pathogen with which it interacts. This has been further refined into a receptor-ligand model, in which each resistance gene encodes a receptor that recognizes the products of avirulence genes in the pathogen (van der Biezen and Jones, 1998). Cell wall-degrading enzymes are used by pathogens to penetrate plant tissue and release nutrients, and among these are the endopolygalacturonases (PGs). These proteins have been shown to be a primary cause of damage caused by several insect species (Girard and Jouanin, 1999; Boyd et al., 2002), fungal pathogens (Kahmann and Basse, 2001), bacteria (Oeser et al., 2002) and possibly nematodes (Jaubert et al., 2002). Pathogens often produce a spectrum of PGs, to allow pathogenesis in various hosts and under diverse conditions (De Lorenzo et al., 2001),

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119 and PGs from different isolates or races of pathogens frequently exhibit polymorphism (Caprari et al., 1993; Daroda et al., 2001; Poinssot et al., 2003). Plants combat these effects in part through polygalacturonase-inhibiting-proteins (PGIPs) (De Lorenzo and Ferrari, 2002). PGIPs not only counteract the ability of PGs to break down cell walls, but also seem to be capable of inducing defense responses (De Lorenzo et al., 2001) by encouraging the formation of long-chain oligogalacturonides. PGIPs are up-regulated in response to stress, wounding, and pathogen attack (De Lorenzo et al., 2001; Yao et al., 1999), and overexpression of PGIPs in transgenic plants have been shown to inhibit fungal colonization (Powell et al., 2000; Ferrari et al., 2003). PGIP proteins possess a Leucine-Rich Repeat (LRR) region, shared by many resistance ( R ) gene products (Martin et al., 2003) and other defense components. In PGIPs, this region is generally ten repeats long, and is the site of much of the diversity in the family. A single amino acid variation in this region has been shown to confer the ability to recognize a specific PG (Leckie et al., 1999) In Arabidopsis, PGIPs are encoded by a pair of adjacent, very similar genes (Ferrari et al., 2003), whereas in bean the family consists of two tandemly-duplicated pairs (D’Ovidio et al., 2004). Mehli et al. (2004) concluded that PGIP genes comprise a small family, of perhaps three to five genes, in the diploid F. vesca genome, as in raspberry (Ramanathan et al., 1997) and apple (Yao et al., 1999). Such gene duplications and divergence within the family may be the result of selection through coevolution with PGs from pathogens. Both Arabidopsis and bean PGIPs have been shown to differ markedly in their abilities to inhibit specific PGs, and in their expression patterns (Ferrari et al., 2003; D’Ovidio et al., 2004).

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120 Mehli et al. (2004) cloned and characterized a F. ananassa PGIP gene, later designated FaPGIP1a. They found it to be highly expressed in red fruit, though there was at least a low level of expression in all other tissues examined. It also appeared to be strongly induced by inoculation with Botrytis cinerea the causal agent of grey mold in strawberry. The five European cultivars used in the study showed varying base levels of expression as well as levels of induction in response to inoculation, although there was not a clear correlation between level of PGIP expression and level of resistance. Later work showed that PGIP was expressed robustly within 8 h of inoculation, and to peak at 16 h after inoculation (Mehli et al., 2005). Schaart et al. (2005) presented a continuation of this study, identifying six distinct alleles of Fragaria PGIP, which they grouped as FaPGIP1, FaPGIP2, and FaPGIP3. All had similar nucleotide and amino acid sequences, and FaPGIP2 and FaPGIP3 appeared similar to F. vesca and F. iinumae respectively, both proposed progenitor species of the octoploid cultivar (Folta and Davis, 2006). Five cultivars were tested for the presence or absence of each of these alleles, with at least two alleles identified in each cultivar. Pyrosequencing identified clear tissuespecific and allele-specific differences in expression, with a majority of expression in the leaf being PGIP2, whereas in the fruit, PGIP1 was the predominant transcript in uninoculated fruit, particularly early in development. In inoculated fruit, however, all three PGIP classes were expressed, in proportion with their representation in genomic DNA (Schaart et al., 2005) PGIPs possess a number of characteristics that suggest they may be a worthwhile target of efforts to develop markers for resistance or to develop resistant transgenic lines. These include the fact that their expression has been shown to be induced by fruit inoculation (Mehli et al., 2004), that their effectiveness has been can be readily altered by small allelic variations, and that bean PGIP proteins have been shown to inhibit the growth of such strawberry pathogens as

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121 Colletotrichum acutatum and B. cinerea (D’Ovidio et al., 2004). The family of PGIP genes in the octoploid also provides an opportunity for comparison with those of diploid species and may cast light on the origins of the octoploid species and on changes in the genome that may have occurred since those species emerged. To explore either of these aspects, it is important to have as complete an accounting as possible of the PGIP genes present in the cultivated strawberry. This report catalogs a suite of Fragaria PGIP genes and illuminates potential utility in disease resistance, as well as their use as beacons for the rapid evolution of the Fragaria genome. Results Identification of Fragaria PGIP Alleles Among the F. ananassa clones, 12 unique PGIP alleles were identified, comprising the three groups defined by Schaart et al. (2005) plus a likely fourth group, that was designated FaPGIP4. Two new alleles of FaPGIP2, designated d and e, and one each of FaPGIP1 and FaPGIP3, designated d and b, respectively, were identified (Table 5-1). Despite extensive sequencing, only one clone of FaPGIP1 was identified from products of the PGIP-seq1 and PGIP-seq2 primers, FaPGIP1d from ‘Treasure’. Because of this, a second forward primer specific to FaPGIP1 sequence (FaPGIP1-F) was used with the same reverse primer and amplified an appropriately-sized fragment in all octoploid and diploid genotypes, suggesting that polymorphism might exist in the portion of sequence matched by PGIP-seq1. Clones generated by this pair from ‘Strawberry Festival’ and ‘Treasure’ were sequenced. Only FaPGIP1b-type clones were found among three sequenced from ‘Strawberry Festival’, however it is possible that these differed from FaPGIP1b at the 5’ end, because this area was omitted with the second primer pair, and no FaPGIP1 were amplified from ‘Strawberry Festival’ using the original set. From ‘Treasure’, the three clones sequenced were either FaPGIP1b, or a previously undocumented allele, FaPGIP1d.

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122 PCR using ‘Strawberry Festival’ cDNA library as template unexpectedly yielded two bands, one of the predicted size based on the mRNA sequence, and another larger fragment of approximately equal abundance, consistent with the genomic sequence. When sequenced this proved to be FaPGIP1a, an apparently unspliced transcript with the intron retained. Although FaPGIP3 type sequences were identified in all the octoploid cultivars in this study except ‘Treasure’, none contained the specific allele previously identified by Schaart et al. (2005). Rather, these cultivars all contain another somewhat different allele, designated FaPGIP3b, which is completely identical to that of F. iinumae within the sequenced region. Such exact correspondence between F. iinumae and an octoploid allele lends additional evidence to the case for F. iinumae as an ancestor of the octoploids. A novel allele type, FaPGIP4, was also identified in ‘Treasure’ and ‘Winter Dawn’. This sequence, though quite similar to the others, did not appear to fit the criteria for any of the established classes in the octoploid, and appears to be more widely diverged from the rest of the family than other alleles. Much of this variation occurs in the coding region, suggesting possible functional differences. Although predicted, functional assessment of these novel alleles remains to be proven, and represents an excellent target for transgenic analysis. Although FaPGIP1-specific primers amplified a product from the diploid species, none were sequenced. Among the clones generated by the original primers, each diploid species yielded only a single type of PGIP allele. The allele from F. vesca ‘Pawtuckaway’ was identical to FaPGIP2c, but did not exactly match any of the three GenBank accessions. However, it most closely resembled the F. vesca A6 allele, differing by a single nucleotide substitution, whereas that sequenced from F. iinumae FRA377 was identical to that in GenBank. PGIPs from F. mandshurica FME and F. bucharica FRA520 were most similar to those of F. vesca and the

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123 PGIP2-type octoploid alleles, though both possessed single nucleotide polymorphisms (SNPs) distinguishing these genes from others. Amino Acid Similarities Among Alleles A majority of the polymorphisms identified did not result in differences in predicted amino acid sequence, and even the most dissimilar, FaPGIP4 and FaPGIP1 or FaPGIP3, shared 90% identity at the amino acid level (Table 5-2). Several of the alleles differed only in intron sequence and yielded identical predicted proteins. Proteins differed at 21 sites within the sequenced region, but fully non-synonymous substitutions occur at only four of these sites, at amino acids 36, 42, 121, and 129, along with an inserted alanine at position 61 in FaPGIP4 and and a deleted threonine in F. mandshurica FME (Figure 5-1). Within the characterized region, the most similar protein from other species was RiPGIP1, from red raspberry (Ramanathan et al., 1997), ranging from 82% (FaPGIP4) to 90% (PGIP2a/b/e, PGIP3a/b, and Pawtuckaway) amino acid identity. Discussion All the genes sequenced were remarkably well conserved. However, because changes in pathogen specificity can occur as a result of a single amino acid difference, it is possible that the sequenced alleles differ in their effectiveness in inhibiting specific pathogen PGs. Most of the variation occurs in the LRR region, which, as the putative site of specificity, is likely to be the portion of the gene under the greatest selective pressure. Most substitutions occurring through natural variation will likely be either neutral or deleterious, with adaptive changes being comparatively rare. In genes undergoing positive selection, the ratio of non-synonymous to synonymous substitutions is generally higher than one, whereas in those undergoing purifying selection or neutral variation the number should be less, often much less. This measure is considered a stringent test of adaptive sequence evolution, but

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124 may not identify cases of weak selective adaptation (Li, 1993).This ratio within the Fragaria PGIPs is slightly greater than one, and is likely indicative of adaptive change occurring within the gene family. Stotz et al. (2000) identified nine amino acid residues in dicot PGIPs for which there was statistical evidence of positive selection, noting that a disproportionate number, though not all, fell into the xxLxLxx beta-sheet regions. Only one of these residues, Ala207 (position based on PvPGIP1 sequence), corresponds to the position of a non-synonymous substitution within the Fragaria PGIPs. FaPGIP1d has an asparagine in this position, whereas all others have aspartic acid. Another non-synonymous substitution, a serine rather than an arginine at position 104 in FaPGIP3a, also occurs within a beta-sheet region. Leckie et al. (1999) identified a particular amino acid in bean PGIP, located at 253 in PvPGIP2, as being critical to the ability to inhibit PG from Fusarium moniliforme In bean, this residue was either a Q or a K, with isoforms possessing the Q capable of recognizing FmPG, and those with the K incapable. Switching this residue to a Q conferred the ability to recognize FmPG on those proteins that were originally incapable, albeit at a relatively low efficiency. Although the strawberry PGIPs differ considerably from their counterparts in P. vulgaris with the most similar pair, F. mandshurica PGIP and PvbPGIP2, sharing only 49% amino acid identity, it is interesting to note that all contain a conserved Q residue in this location. Because Schaart et al. (2005) had shown the FaPGIP2 and FaPGIP3 genes to be similar to F. vesca and F. iinumae respectively, it was anticipated that the FaPGIP1 sequences might match one of the other diploid species suggested as ancestors of the octoploid species. Potter et al. (2000), using both genomic and chloroplast sequence, identified F. nubicola and F. orientalis as belonging to the same clade as the cultivated octoploid, whereas DiMeglio and Davis

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125 (unpublished but discussed in Folta and Davis, 2006) suggested that F. mandshurica (possibly a diploid form of F. orientalis ) was a candidate. Yet the sequence from F. mandshurica did not match any of the other sequenced alleles, differing from the octoploid sequences at numerous sites, including a 3bp deletion in the coding region. Although the accession intended as F. nubicola was later identified as F. bucharica it is in fact the same accession used by both Potter et al. (2000) and DiMeglio and Davis and thus their suggestions should remain valid. However, while resembling both the F. vesca alleles and the FaPGIP2 octoploid genes, the F. bucharica sequence did not exactly match any of them. It seems likely that although all the genes examined here are closely related, not all the classes of octoploid alleles represent homoeologous loci, duplicated through polyploidy. Given that PGIP genes have been shown to be tandemly-repeated in nearly every genome in which it has been investigated, including the closely related raspberry (Ramanathan et al., 1997; D’Ovidio et al., 2004; Ferrari et al., 2003), it is not unreasonable to suppose that two or more or more of the Fragaria genes described here may represent such a duplication. The FaPGIP2 and FaPGIP3 alleles are more similar than the other two classes, and the close parallels within diploid genomes strongly suggest that these may represent the same locus in different octoploid genomes. Yet FaPGIP1-type genes appear to exist in these diploid species in addition to FaPGIP2 or FaPGIP3-type genes, so this may represent a separate locus. Whether or not the different FaPGIP1 alleles obtained from the octoploid represent variation at a locus within a single genome or in multiple genomes is difficult to ascertain. There is more variation in the FaPGIP1 genes than in the FaPGIP2 or FaPGIP3 genes, but they are more similar to each other than these two classes are to each other. The FaPGIP1b allele appears to occur in all the European cultivars and all the American cultivars surveyed here except ‘Treasure’, and may

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126 represent a non-polymorphic locus within a specific genome that is not recombining with those that contain the other alleles. The FaPGIP1d form present in ‘Treasure’ differs from FaPGIPb by only a single nucleotide. It is also possible that some of the of the FaPGIP1-type alleles have been missed by this survey because of variation at one or both of the primer sites. In the previous study of European octoploids (Schaart et al., 2005), only a single FaPGIP3 allele was identified, and this was found in three of the five European cultivars. A single different FaPGIP3 allele was identified in most of the American cultivars. This suggests a lack of diversity at this locus, and a clear split between the European and American cultivars. Although European octoploids are derived from New World species, European and American breeding programs have engaged in comparatively little exchange of germplasm, as the different environmental conditions require material with different adaptations, and in this case the two groups represent regions of very different climates: European cultivars from Northern and Eastern Europe, and American cultivars from Florida and California. Methods and Materials Plant and Genetic Material A wide range of cultivated and wild germplasm was screened for PGIP alleles. DNA was extracted from young leaves of the F. ananassa cultivars Strawberry Festival, Winter Dawn, and Treasure using the Qiagen DNEasy Plant Mini Kit, according to the manufacturers instructions. Additionally, Dr. Thomas Davis of the University of New Hampshire provided genomic DNA from F. vesca ‘Pawtuckaway’, F. bucharica FRA520 (originally considered F. nubicola but reclassified by Staudt (2003)), F. iinumae FRA377, and F. mandshurica FME, and Dr. Kimberly Lewers of the USDA Agricultural Research Service in Beltsville, Maryland provided genomic DNA of the F. ananassa cultivar Selva. A cDNA library derived from all

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127 plant tissues of salicylate-treated ‘Strawberry Festival’ (described in Folta et al., 2005) was also used as template. Cloning and Sequencing of Fragaria PGIP Alleles Oligonucleotide primers were designed based on regions of consensus sequence within four diploid Fragaria PGIP sequences available on GenBank: AF196893, AF196892, and AF196891 from several selections of F. vesca and AF196890 from F. iinumae These primers, FrPGIP-F (5’-CTCATGGAAATCCGACGCCG), and FrPGIP-R, (5’CGACAAGCTTGATCTCACTG) flanked the entire coding region. Because early sequencing and later publications by Mehli et al. (2004) and Schaart et al. (2005) showed that there was little or no variation outside of the intron and LRR region, some PGIP alleles were amplified using primers published by Schaart et al. (2005) that flanked solely these regions: PGIP-seq1 (5'CCTCCATCGCCAAGCTCAAG), and PGIP-seq2 (5'-CAAGTCCAGTGAGATCAAGC). A FaPGIP1 specific forward primer FaPGIP1-F (5’-CAACGAATTTCCCGTATGCGCG) was also used separately, in conjunction with the PGIP-seq2 reverse. The resulting amplicon from each genotype was cloned into the pCR2100vector, transformed into E. coli and purified using the alkaline lysis plasmid preparation protocol (Bringborm and Doly, 1983). Samples were then quantified via spectrophotometry and submitted for sequencing from the M13 forward and reverse primers. A total of 54 clones were sequenced. Sequence Analysis The genomic sequences were aligned using Clustal W software hosted by European Molecular Biology Laboratory – European Bioinformatics Institute (EMBL-EBI) at http://www.ebi.ac.uk/clustalw/, using the default settings. Phylogenetic trees were developed utilizing this software, using the neighbor joining (NJ) method of Saitou and Nei (1987). Nucleotide sequences were translated into amino acids using the Molecular Toolkit software

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128 (http://arbl.cvmbs.colostate.edu/molkit/translate/index.html), hosted by Colorado State University, and splice sites identified according to the mRNA sequence provided in Mehli et al. (2004).

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129 Table 5-1. FaPGIP alleles identified in various octoploid strawberry cultivars. FaPGIP isoform Cultivar1a1b1c1d2a2b2c2d2e3a3b4 Elsantaz+++ Koronaz++++ Polkaz+++++ Senga Senganaz++++++ Teniraz++ Strawberry Festival++++ Winter Dawn+++++ Treasure+++ Selva++++ z European cultivars from Schaart et al. (2005) presented here for comparison purposes.

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130 Table 5-2. Percentage of identity between PGIP isoforms at the mRNA (standard print) and amino acid (bold face) levels.1a1b1c1d2a2b2c2d2e3a3b4FvFiFbFmRi1 FaPGIP1a—99999798989897979695959895969686 FaPGIP1b 100 —999998989898989697959897989788 FaPGIP1c 9999 —9998989898989696959896989787 FaPGIP1d 989897 —98989998989696969896989786 FaPGIP2a 98989797 —10099999997989510098999888 FaPGIP2b 98989797100 —99999997989510098999888 FaPGIP2c 979797969999 —99999798969998999888 FaPGIP2d 97979796979798 —999798969998999988 FaPGIP2e 989897971001009997 —9798969998999988 FaPGIP3a 969695959797979797 —99949799979688 FaPGIP3b 97979796989897979897 —9598100989788 FaPGIP4 9090908992929191929090 —9595969585 FvPGIP 989897971001009997100979892 —98999888 FiPGIP 979797969898979798971009098 —989788 FbPGIP 9797969598989797989697909897 —9987 FmPGIP 979797969999989899959792999797 —87 RiPGIP1 88888888909089899090908290908889 —

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131 FaPGIP1a L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPNSLSKLPNL LALHLDRNQLT 60 FaPGIP1c L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPNSLSKLPNL LALHLDRNQLT 60 FaPGIP2a L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPSSLSKLPNL LALHLDRNQLT 60 FaPGIP2c L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPSSLSKLPNL LALHLDRNQLT 60 FaPGIP3 L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPSSLSKLPNL LALHLDRNQLT 60 FaPGIP4 L KMLRFSWNGLCGSFPDFLSQLKNL TFLELNYTNFTGSVPSSLSKVPNW LALHLDRNQLT 60 FbPGIP L KMLRLSWNGLSGSVPDFLSQLENL TFLELNYNNFTGSVPSSPSKLPNL LALHLDRNQLT 60 FiPGIP L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPGSLSKLPNL LALHLDRNQLT 60 FmPGIP L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPSSLSKLPNL LALHLDRNQLT 60 FvPGIP L KMLRLSWNGLSGSVPDFLSQLKNL TFLELNYNNFTGSVPSSLSKLPNL LALHLDRNQLT 60 *****:*****.**.*******:*********.*******.* **:** *********** FaPGIP1a -GNIPSAYGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRIDLSRNMLEGDASMIFGMNK 119 FaPGIP1c -GNIPSAYGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRLDLSRNMLEGDASMIFGMNK 119 FaPGIP2a -GNIPSSYGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRIDLSRNMLEGDASMIFGMNK 119 FaPGIP2c -GNIPSSYGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRIDVSRNMLEGDASMIFGMNK 119 FaPGIP3 -GNIPSSFGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DQIDLSSNMLEGDASMIFGMNK 119 FaPGIP4 AGNIPSSYGKLVGTV PDLFLSHNKLTGKIPTSFGNMNF DRIDLSRNMVEGDASMIFGMNK 120 FbPGIP -GNIPSSYGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRIDLSRNMLEGDASMIFGMNK 119 FiPGIP -GNIPSSFGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRIDLSRNMLEGDASMIFGMNK 119 FmPGIP -GNIPSSYGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRIDLSRNMLEGDASMIFGMKK 119 FvPGIP -GNIPSSYGKFVGTV PDLFLSHNKLTGKIPTSFANMNF DRIDLSRNMLEGDASMIFGMNK 119 .*****::**:**********************.*****::*:* **:**********:* FaPGIP1a TT QIVDLSRNMLEFDLSKVVFST 142 FaPGIP1c TT QIVDLSRNMLEFDLSKVVFST 142 FaPGIP2a TT QIVDLSRNMLEFDLSKVVFST 142 FaPGIP2c TT QIVDLSRNMLEFDLSKVVFST 142 FaPGIP3 TT QIVDLSRNMLEFDLSKVVFST 142 FaPGIP4 TA QIVDLSRIMLEFDLSKVVFST 143 FbPGIP TT QIVDLSRNMLEFDLSKVVFST 142 FiPGIP TT QIVDLSRNMLEFDLSKVVFST 142 FmPGIP TQIVDLSRNMLEFDLSKVVFST 141 FvPGIP TT QIVDLSRNMLEFDLSKVVFST 142 ******* *************Figure 5-1. Amino acid alignment of the polymorphic portion of a number of Fragaria PGIP genes. Shaded areas indicate the xxLxLxx beta-sheet regions. Note that several alleles are omitted because they encode identical proteins to others listed despite differing nucleotide sequences.

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151 BIOGRAPHICAL SKETCH Philip J. Stewart was born in Olean, New York, on July 30, 1974. He received his elementary and secondary education at Cuba-Rushford Central School, Cuba, New York, graduating in 1992. Upon graduating, he received a National Merit scholarship to Alfred University, in Alfred, New York, where he obtained a Bachelor of Arts in Biology in 1996. He accepted a job as a research technician with Cornell University, at the New York State Agricultural Experiment Station in Geneva, New York. He spent five years in this position, assisting in the development of wine and table grape cultivars for the Finger Lakes Region and conducting research into bud cold hardiness and grape pedigrees. In August, 2000, he married Cynthia R. Cowan, in Rochester, New York, and soon returned to school to further study fruit breeding. In 2003 he received a Masters of Science in Horticulture from the University of Arkansas, where he studied disease resistance in blackberries under Dr. John Clark. He began a doctoral program at the University of Florida starting in January of 2004, investigating the molecular biology of strawberries with Kevin Folta and Craig Chandler. During the course of his studies, he and his wife welcomed two daughters Zea, in 2002, and Adina in 2006. Philip has accepted a job as Strawberry Breeding Manager with Driscoll Strawberry Associates, developing proprietary strawberry varieties for Driscoll’s growers in a number of regions of North America. He will begin employment in Watsonville, California upon completion of his degree.


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MOLECULAR CHARACTERIZATION OF PHOTOPERIODIC FLOWERING IN
STRAWBERRY (Fragaria SP.)



















By

PHILIP JACOB STEWART


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA INT PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

May 2007

































02007 Philip J. Stewart























To my parents,
who always believed in me, and taught me to love knowledge and the natural world.

To the memory of my grandfather, Jackson Lee Stewart, Jr.,
who introduced me to agriculture, and who will forever be my image of the American farmer.

To my children, Zea and Adina,
who have been a constant inspiration and joy to me,
and reminders of the things in life that are truly important.

And, most of all, to my wife, Cynthia,
who has been unwavering in her faith in my abilities,
in her willingness to sacrifice for my academic and professional pursuits,
and, most importantly, in her love.









ACKNOWLEDGMENT S

I would like to take this opportunity to thank at least a few of the many people who have

have assisted me along the path to this degree.

First, I would like to offer my love and gratitude to my parents, Lee and Faith Stewart,

and my in-laws, Barton and Teri Cowan, all of whom have provided continuous support, both

emotional and Einancial, throughout the course of my graduate studies. None of this would have

been possible without them.

Secondly, I would like to thank those that who have provided invaluable assistance in my

academic and professional development, in particular my graduate advisors, Kevin Folta and

Craig Chandler. Their help was critical to the work described here. I am also grateful for the

assistance and suggestions offered by the other members of my committee: Paul Lyrene, Natalia

Peres, and Jose Chaparro, all of whom helped to shape this study, and Daniel Sargent, of East

Malling Research, U.K., for the use of his diploid mapping population. Much thanks also go to

Dawn Bies and Maureen Clancy, for technical assistance, and also to my fellow students in the

Folta lab: Denise Tombolato, Stefanie Maruhnich, Thelma Madzima, and Wendy Gonzalez. I've

enjoyed working with all of you and wish you much success as you Einish your studies and move

into professional life.

And, Einally, the greatest debt of gratitude is owed my beautiful wife, Cynthia, who has

not only been tolerant but encouraging as I have dragged her from place to place across the

country in pursuit of this dream. Thank you so much--I hope it was worth it.











TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ................ ................. 4...............

LI ST OF T ABLE S................................ 8

LI ST OF FIGURE S ................. ................. 9......... ...

AB STRAC T ................. ................. 1......... 1....

CHAPTER

1 INTRODUCTION ................. ................. 12.............

2 LITERATURE REVIEW.............. ................. 14


The genus Fragaria .............. .............. 14..._ _. ....
Polyploidy in Strawberries .........._.... ............... 15...__ .. ....
Flowering Habits in Strawberry ........._..... ............... 16....._. ...
Short-Day Strawberries .........._.... ............... 16..._._. .....
Remontant Strawberries .........._.... .... .. ... ............... 18....
Factors Affecting Expression Of Photoperiodic Flowering ................ ................. ...._23
Temperature Effects On Flowering ................. ................. 23.............
Vernalization Requirements .........._.._ ......... ............... 24....
Juvenility and Plant Age Effects. .........._.._......... ...._ .... ....._._ .............2
Growth Pattern Differences Between Plants Of Differing Flowering Habits .........._.._..........26
Inheritance Of Flowering Habit In Strawberry .........._.._. .....__ ........__..........2
Molecular Markers For Flowering Habit ......_.._._ .... ..._.. .....___ ..........3
Molecular Control Of Flowering In Arabidopsis ................ ..............33. .............
Components Upstream of CO. ................ ................. 34.............
Components Downstream of CO ................ ..............35. ..............
The CONSTANS-LIKE Gene Family ..........._. .. .. ....... .. ...... ...........3
Components of the Flowering Pathways are Conserved Among Species .........._.........38
Using Model Systems To Understand Development In Rosaceae .........._... .............40

3 CHARACTERIZATION OF CONSTANS-LIKE GENES IN Fragaria .............. .............44

Introduction ............_... ............... 44..._._. ....
R results ........._... ... ......... ._... .... ... ._ _... .................. .. .......4
Cloning And Identification Of Four CONSTANS-LIKE Genes Of Fragaria.............__46
FrCO/FrCOL Gene Structure ........._._._....... .... ............... 48...
Southern blot analysis of FrCO Copy Number ..................... .._.. ..... ......... .... 50
Mapping Of Fragaria CO And COL Genes In A Diploid Mapping Population ........... 50
Comparison Of Strawberry Genotypes Under Varying Photoperiods ................... ........ 51
Allele-Specific Expression Patterns Differ Betwen Short-Day And Day-Neutral
Genotypes ................ ................. 52..............












Expression Of Other Genes Under Different Photoperiods ................. ............... .... 53
Some FraCOL2 Transcripts Contain An Unspliced Intron.............. ................. 54
Discussion .............. ... ....... _. .... .. .............. 54
The Co Gene Family In Fragaria (Rosaceae) ......._.__ ..... ...._ .. ....._.._.......5
Evolutionary Relationships Among FrCO Sequences.............. ............... 58
FrCO Expression ........._ ....... .... ..............61....
Expression of Other COL Genes ........._.. ....___......... ...........6
Materials and Methods ........._ ........_. ..............68....
Plant M material ............_._ ..............68._.._......
Plant Growth Conditions ........._ ........_. ..............69....
Cloning of FraCO .........._... ... ..... .. ..............70...
Identification of COL Genes in Genebank.............. ................70
Gene Structure Characterization.............. ............ 70
Southern Blot Analysis ........._.._.._ ......_. ..............71.....
Genetic Linkage Mapping COL genes in Fragaria ......____ .......___ .............71
Extraction of Nucleic Acids ........._.__........_. ..............72....
Reverse Transcription and RT- PCR ............_. .....___ .....__ ...........7

4 IDENTIFICATION AND CHARAC TERIZATION OF FLOWERING-RELATED
GENES IN Fragaria AND OTHER ROSACEAE ................. ................. ......... ..93

Introduction ................. ................. 93.............
Results ................. ...... ...... .... ......... .. ... ..........9
Identification of Rosaceae Flowering Gene Homologs ................. ........... .......... 94
FrSOC1 Expression ................. ................. 97.............
FrTFL1 and FrLFY Expression ........._.. ....___........_. ...........9
Discussion ........._...... .... .... .............. 98....
Rosaceae MADS-Box Genes ............_._ ..............98..._. ....
The FT/TFL1 Gene Family in Rosaceae.............. ............... 101
Other Flowering Genes ........._ ........_. ............._ 105..
FrSOC1, FrLFY, and FrTFL1 Expression ..............._ ................ 105......._...
Methods and Materials................ ...... ......... 109
Identification of Rosaceae Flowering Genes ..............._ .............. ........_.... 109
Phylogenetic Tree Construction ................. ......... ......... .. ..... ....10
RNA Extraction and RT-PCR Experiments ................ .............................. 109


5 Fragaria ALLELES OF POLYGALACTURONASE INHIBITOR PROTEIN GENES... 118


Introduction ............. ...... __ ............._ 118...
Results .............. ........__ ........ ........ ..........12
Identification of Fragaria PGIP Alleles .................._ ....__ ..........12
Amino Acid Similarities Among Alleles ........._ ....... __ ....._ .......... 2
Discussion ............. ..... .. ............. 123...
Methods and Materials.............. ............... 126
Plant and Genetic Material ............. ............._ ....._ .......... 12
Cloning and Sequencing of Fragaria PGIP Alleles ............_. ....... ............ 127
Sequence Analysis ........._ ........_. ............._ 127...












LIST OF REFERENCES .........._.._ ......... ............._. 132...


B IO GRAPHIC AL SKET CH ........._.._.. ...._... ............... 151










LIST OF TABLES


Table page

3-1 Comparison of amino acid identity (%) of the predicted protein encoded by FraCO to
those of Group la CONSTANS-LIKE genes in various species. .........._. .........._.....75

3-2 RT-PCR primers for strawberry CONSTANS-like genes and controls, sequence
source, Thl, and approximate size in cDNA (as calculated from sequence). ................... .76

3-3. GenBank accession numbers for Fragaria COL genes and other Rosaceae orthologs....77

3-4 Mean inflorescence and runner production of. xanana~ssa and E. vesca genotypes
under LD (16 h) and SD (8 h) photoperiods, at 18/160C day/night temperature. ............ 86

3-5. Sources of plants used in this chapter. ................ .......... .. .............. 90.

4-1 Identified Fragaria genes related to flowering and photoperiodic response, with the
closest match at the amino acid level among Arabidopsis and all annotated
transcripts. ................ ................ 112........ .....

4-2 RT-PCR primers for strawberry flowering genes and controls, sequence source, Thl,
and approximate size in cDNA (as calculated from sequence). ................ ............... 114

5-1 FaPGIP alleles identified in various octoploid strawberry cultivars. ............. ............ 129

5-2 Percentage of identity between PGIP isoforms at the mRNA and amino acid levels..... 130










LIST OF FIGURES


Figure page

2-1 Simplified diagram showing interactions of genes and environmental factors
governing flowering in Arabidopsis thalianaiii~~~~~iiiii~~~~ ....._.. ............_. ..........__....... 4

3-1 Cladogram of CO-like genes, including all Arabidopsis and full-length Rosaceae
COL genes as well as all those from other species Group la genes demonstrated to
functionally complement Arabidopsis co mutants. ................ ................ ......... 78

3-2 Alignment of the predicted amino acid sequence for FraCO with those of AtCO and
functionally confirmed dicot homologs, with maj or conserved domains noted. ........._.....79

3-3 Alignment of the predicted amino acid sequence for FrvCOL1 with possible apple
and Arabidopsis homologs, with maj or conserved domains noted. ................ .............. 80

3-4 Structures of COL alleles from Fragaria species: xanana~ssa Strawberry Festival'
(Fra), E. vesca FDP815 (Fry), E. nubicola FDP 601 (Frn), and E. iinumae FRA377
(Fri) ................. ................. 8......... 1....

3-5 NJ phylogram tree of intron sequence divergence among FrCO alleles..............._.._. ..... 82

3-6 Comparisons of hypervariable section of coding region in FrCO. ........._.._.. ........._.... 83

3-7 Southern blot of genomic DNA from E. xanana~ssa 'Strawberry Festival' and F.
vesca Hawaii 4, cut with six different enzymes and probed with a portion ofFrvCO ..... 84

3-8 Mapping of FrCO in a reference diploid population. ........._._......___ ............. 85

3-9 Northern blot of RNA from Strawberry Festival', a SD genotype, and 'Diamante', a
DN genotype, collected every 4 h, showing expression of FraCO under short and
long day conditions. ........... _.. .............. 87..._ _. ....

3-10 RT-PCR assay of expression ofFrCO transcript under short (8 h) and long (16 h)
days in three genotypes: E. vesca Hawaii-4 (SD, diploid), xanana~ssa 'Camarosa'
(SD, octoploid), and xanana~ssa 'Diamante' (DN, octoploid). ............. .................. 88

3-11 Expression of 35 and C9 alleles ofFraCO at dawn under SD, using RT-PCR at 33
and 40 cycles, in 'Strawberry Festival' (F) and 'Diamante' (D). ................ ................ 89

3-12 RT-PCR assay of FrCOL2 expression in E. vesca Hawaii-4, showing variation in
splicing efficiency at several points under short days. ......... ................ ..............91

3-13 Ubiquitin controls after equalization of RT-PCR samples using RN-Ubiql primer set
to standardize template amounts. ........._.. ...._._ ..............92...










4-1 NJ tree showing similarity between full-length amino acid sequences of Rosaceae
and Arabidopsis MADS-Box genes ................ ................ 111........ ....

4-2 NJ phylogeny tree showing relationships between Arabidopsis and Rosaceae
members of the FT/TFL1 gene family, based on full predicted protein sequence. ........ 1 13

4-3 RT-PCR assay of expression of FrSOC1 under short (8 h) and long (16 h)
photoperiods in 'Camarosa' (SD, octoploid) and 'Diamante' (DN, octoploid). ........... 115

4-4 RT-PCR assay of expression of FrLFY under short (8 h) and long (16 h)
photoperiods in Hawaii-4 (SD, diploid) and 'Camarosa' (SD, octoploid). ................... 116

4-5 RT-PCR assay of expression ofFrTFL~ under short (8 h) and long (16 h)
photperiods in Hawaii-4 (SD, diploid). ................ ......... ......... ..........17

5-1 Amino acid alignment of the polymorphic portion of a number of Fragaria PGIP
genes. Shaded areas indicate the xxLxLxx beta-sheet regions. ........._.__... ......_...... 131









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MOLECULAR CHARACTERIZATION OF PHOTOPERIOD FLOWERING IN
STRAWBERRY (Fragaria)

By

Philip J. Stewart

May 2007

Chair: Craig Chandler
Cochair: Kevin Folta
Major: Horticultural Science

The initiation of flowering is a critical developmental transition for most plant species,

affecting reproductive potential and evolutionary success. The timing of this event in strawberry

(Fragaria sp.) is conditioned by a number of factors, including photoperiod. Sensitivity to

photoperiod varies among strawberry genotypes, which are generally divided into three

categories: short-day, everbearing, and day-neutral. The genetic basis for these variations is not

known, but existing model plant systems such Arabidopsis thaliana and rice reveal a well-

conserved network of genes and proteins governing the perception of photoperiod and the

regulation of floral initiation. A number of these genes were identified and characterized in

strawberry and other relatives in the family Rosaceae, and their expression in strawberry under

long and short photoperiods assayed. Expression profiles suggest that while many of the critical

genes governing photoperiodic flowering are conserved between strawberry and model species,

their expression and relationships to one another are unlike those of any previously characterized

plant species.









CHAPTER 1
INTTRODUCTION

The initiation of flowering is among the most important developmental transitions a plant

makes, second perhaps only to germination. Mistimed flowering endangers the reproductive

potential of plant in a number of ways. A plant that flowers too early or too late may find the

seasonal conditions not conducive to seed development, or yield seeds that germinate at points in

the season that make seedling survival difficult or impossible. In cross-pollinated species even a

difference of a few days may be adequate to render reproduction impossible, putting a plant out

of syncronization with others of its species. Any of these difficulties has the potential to render

an individual an evolutionary dead end.

Because of this necessity, plants have developed an extraordinary regulatory network

allowing the Eine tuning of the transition to flowering by a range of environmental and internal

factors. In the model eudicot Arabidopsis, these factors have been shown to include the

environmental inputs of photoperiod, light quality, and temperature, and internal pathways

controlled by the hormone gibberellic acid and autonomous factors such as developmental age.

At the molecular level, each of these inputs represents separate elements of genetic

pathways that converge at a few floral integrators to regulate flowering through a common set of

meristem identity genes. These integrators convert the complex and heterogeneous inputs from

the various environmental and internal cues into a decision to flower, influencing a choice made

in a small cluster of meristmatic cells, and driving development from a vegetative to

reproductive state through the activation of genes that remodel cell fate. Key among these floral

integrators is CONSTANS (CO), which serves to tie the perception of photoperiod with that of

the spectral quality of light received.










Flowering in cultivated strawberry (Fragaria xanana~ssa Duch.) has historically received

a great deal of attention from researchers, for two primary reasons. First, strawberry is an

attractive platform for flowering research because despite a narrow genetic base (Sjulin & Dale,

1987) it exhibits at least three distinct and ranging flowering habits: junebearing (short day), day-

neutral (photoperiod insensitive), and everbearing (often referred to as "long day" but more

accurately another distinct form of photoperiod insensitivity). These clear phenotypic

delineations in such a genetically homogeneous background suggests fairly simple genetic

difference between markedly contrasting flowering habits.

Secondly, in an increasingly global market where fruit is readily transported not only

across countries but around the world, farmers depend on a precise understanding of flowering

behavior to time harvest to coincide with the best market windows in order to obtain the best

price for their fruit. In an era of narrowing profit margins, this timing may be the difference

between a profitable crop and a loss.









CHAPTER 2
LITERATURE REVIEW

The genus Fragania

All strawberry species belong to the genus Fragaria, and are members of Rosaceae, a

family that contains a large number of economically significant crops, primarily fruits such as

apple (Malus domestic Borkh.), pear (Pyrus sp.), stone fruits (Prunus sp.), and brambles (Rubus

sp.), but also ornamentals such as rose (Rosa sp.). Strawberries belong to the Rosoideae

subfamily, which includes the genera Rubus, Rosa, Potentilla, and Duchesnea. Authorities vary

somewhat on the exact number of strawberry species, but most recent authors list about twenty

species (Hancock and Luby, 1993; Folta and Davis, 2006). Native strawberries occur in much of

North America, Europe, and Asia, as well as parts of South America, and Hawaii (Darrow, 1966;

Hancock and Luby, 1993).

Cultivated strawberry, xanana~ssa Duch., is a species of relatively recent hybrid origin,

being derived from the accidental hybridization of two New World octoploid species, F.

virginiana Duch. and E. chiloensis (L.) P. Mill., beginning in France some time in the late 1700s

(Darrow, 1966). Although the polyploid nature of the crop and its hybrid origin have assured that

the species contains some genetic diversity, the actual germplasm base used is relatively small,

with only 53 founding clones (Sjulin and Dale, 1987) and 17 initial cytoplasm donors (Dale and

Sjulin, 1990) contributing to the cultivated varieties. Some recent introgressions from the wild

have been made, most notably from a selection of E virginiana ssp. Duch. var. glauca (S. Wats)

Staudt, but in general very little new genetic material has been added since the early years of

strawberry breeding (Darrow, 1966).









Polyploidy in Strawberries

As has been previously mentioned, the cultivated strawberry is an octoploid (2n=8x=56).

The maj ority of strawberry species, however, are diploids, and there exist tetraploid and

hexaploid taxa as well (Folta and Davis, 2006). Considerable evidence suggests that the

cultivated strawberry is an alloploid, with either three or four distinct subgenome types. The

earliest model, that of Federova (1946), proposed a composition of AABBBBCC, but this was

replaced with AAA'A'BBBB when Senanayake and Bringhurst (1967) found evidence of partial

homology between the A and C genomes. Mounting evidence of diploidization (Bymne and

Jelenkovic, 1976; Arulsekar, et al. 1981) prompted Bringhurst (1990) to propose a fully

diploidized model: AAA'A'BBB'B'.

The origins of the component genomes have long been a subj ect of debate. As early as

1926, Ichijima suggested that one of these might be derived from E. vesca, based on cytogenetic

observations ofE. virginiana (8x) x E. vesca L. (2x) hybrids, in which the formation of seven

bivalents was noted. Further data, including cytology (Senanayake and Bringhurst, 1967) and

sequence analysis of the chloroplast trnL-tmnF region (Potter et al., 2000), the nuclear sequences

ITS (Potter et al., 2000) and polygalacturonase inhibitor protein (PGIP) (Chapter 5 of this work),

have strengthened this view, and the A genome is now widely believed to be derived from F.

vesca (Folta and Davis, 2006).

The origins of the other genomes have been less clear. The study of ITS and tmnL-trnF

sequences implicated E. nubicola as a possible A-type genome donor, in addition to E. vesca

(Potter et al., 2000). Recent work by DiMeglio and Davis (unpublished but cited in Folta and

Davis, 2006) examining the alcohol dehydrogenase locus implicates two other species, F.

mandshurica Staudt and E. iinumae Makino. Potter et al. (2000) and Harrison et al. (1997) found










E. iinumae to be the most distantly related of all other Fragaria species examined, and as such

may represent the ancestor of one or both of B and B' genomes. Unlike most diploid species,

which possess at least a degree of compatibility with other diploid Fragaria (Dowrick and

Williams 1959), E. iinumae is not compatible with E. vesca (Bors, 2000; Sargent et al., 2004b),

and displays a karyotype distinct from the other diploid species (Iwatsubo and Naruhashi, 1989,

1991), which may account for the failure of B genome chromosomes to pair with those of the A

genomes. E. mandshurica, on the other hand, is a newly described diploid species (Staudt, 2003)

not included in previous studies. It is however thought to be related to the tetraploid species F.

orientalis Losinsk., to which it bears a strong resemblance. Potter et al. (2000) had placed F.

orientalis in the A clade, and thus E. mandshurica may represent A' genome in the octoploid

species.

Flowering Habits in Strawberry

Although many authors (Fuller, 1897; Fletcher, 1917) had suggested the presence of

environmental influences on flowering in strawberry, Sudds (1928) was perhaps the first to note

the specific effects of photoperiod and temperature on flower bud induction in strawberry. Soon

after, Darrow and Waldo (1929, 1930, 1933) conducted an exhaustive series of experiments,

classifying over a hundred octoploid strawberry genotypes as flowering under either short or

long days, finding the groups to generally coincide with what were already termed "June

bearers" and "ever bearers".

Short-Day Strawberries

It is believed that the natural state of most strawberry species is as a short day (SD) plant

(Darrow, 1966), though there is considerable variation in sensitivity. It appears that the earliest

cultivated octoploids all flowered under short days (Darrow, 1966), although the limits of the

required photoperiod vary considerably between genotypes. In general, most SD genotypes form









flower buds under photoperiods of less than 14 h, which was first suggested by Darrow (1936).

Three maj or factors seem to dictate response to photoperiod: genotype, temperature, and chilling.

Genotype effects on photoperiodic flowering in SD strawberries

Darrow concluded that photoperiods of 9.5 to 13.5 h induced the greatest number of

flowers in SD cultivars, with an optimum around 12 h (1936). While still generally true, further

studies and perhaps continued progress in breeding have widened this range somewhat. On one

end of the range, some authors (Izhar, 1997; Faedi et al., 2002) have delineated a class of

strawberries they refer to as "infra short-day." These plants flower under longer day lengths

(typically 13.5-14 h photoperiod) and with little chilling requirement, but seem to represent

merely an extension of the existing spectrum of sensitivities already known in SD plants, rather

than a distinct flowering habit. In other genotypes, photoperiods of considerably less than 12 h

are required for optimal flowering. In a study by Heide (1977) 'Abundance' demonstrated peak

flowering under a 10 h photoperiod, the shortest tested in the study. A number of studies have

shown robust flowering under 8 h photoperiods in some cultivars (Sonsteby, 1997; Leshem and

Koller, 1964; Barger et al. 1997; Moore and Hough, 1962). Many early researchers compared

short and long day lengths in terms of natural light versus natural light plus an artificial extension

of day length, making it difficult to assign specific photoperiods.

It is interesting to note that while SD photoperiods are needed for optimal floral

induction, they may actually delay floral development. Several studies of SD cultivars have

shown that continuing SD photoperiods may actually slow the development of previously

initiated buds compared to longer photoperiods (Moore and Hough, 1962; Durner and Poling,

1987). Consequently, Salisbury and Ross (1992) consider strawberries to be SD for purposes of

flower induction, but LD plants for flower development. A similar relationship for temperature

has also been noted (see below).









Remontant Strawberries

Alpine strawberries

The first maj or exception to this flowering pattern noted was the alpine strawberry, F.

vesca var. semperflorens Duch. Found at several places in the European Alps, these represent a

mutation of the common diploid European wood strawberry, and were among the earliest types

to be cultivated. Though first mentioned as early as 1553, they gained prominence in the mid

1700's when they were introduced into France and England, possibly from Turin, Italy

(Duchesne, 1766). In 1811, a runnerless form was obtained by Labaute (Darrow, 1966). Now

only occasionally cultivated, these varieties are of only minor commercial importance (Darrow,

1966), although in the early nineteenth century a white-fruited variety was extensively cultivated

in Quebec (Fletcher, 1917). Fairly little work has been done to characterize the floral physiology

of these varieties, and the fact that many workers have studied unnamed genotypes that are either

seed-propagated or even collected directly from the wild (Chabot, 1978) makes it difficult to

consistently track genotype-specific differences. Sironval and El Tannir-Lomba (1960) found

that their selection ofF. vesca var. semperflorens required days longer than 12 h to form flower

buds, and that SD treatments inhibited flowering in plants that had previously reached the

flowering stage under LD conditions, though this appears to be the exception, not the rule

(Darrow, 1966). As in other strawberries, temperature has a significant effect, with moderately

low temperatures favoring both reproductive development and biomass production (Chabot,

1978).

"Everbearers"

Relatively quickly after the introduction of octoploid cultivars, however, so-called

everbearingg" (EB) strawberries were identified (Darrow, 1966). Such genotypes initiate

flowering most heavily under the long days of summer, and often on unrooted or newly rooted









runners, resulting in what is in most locations primarily a fall harvest, rather than an early

summer one, or in some cases two distinct crops.

These genotypes appear to trace to a few distinct sources within cultivated types.

suggesting spontaneous mutation of a single gene. Development of such types took place

independently in both North American and Europe, but very little crossover has ever occurred

between these two sources of the trait. The first North American EB cultivar described was

possibly 'Oregon Everbearing,' obtained in 1882, although Fletcher (1917) describes an

everbearing selection of what was likely E. virginiana, found in Ohio in 1852. The first

successful everbearer, however, was likely 'Pan-American,' an apparent sport of the June bearer

'Bismarck' found in 1898 (Fletcher, 1917). 'Pan-American,' in turn, became the source of the

trait for a number of other successful everbearing cultivars, including 'Progressive' and

'Rockhill' (Darrow, 1966). This last selection is the source of the trait in the proprietary

everbearing cultivars developed by Driscoll Strawberry Associates (Darrow, 1966). Other

sources of the trait in North America include 'Gem,' a sport of 'Champion' introduced in 1933

that proved quite popular for several decades. That the trait is the result of instability at a single

locus is supported by documented reversion to short day flowering in the case of 'June Rockhill',

a short day mutant of the everbearer 'Rockhill' (Darrow, 1966).

European octoploid everbearers, called "remontant" by the French, may have had their

origin somewhat earlier. Unlike the American sources of the trait, which can largely be traced to

mutations identified in cultivated plants, the source of the European everbearers is unclear.

Although claimed at the time to be hybrids of large-fruited octoploids with the everbearing

Alpine diploids (Fletcher, 1917), it seems doubtful that this was truly the case. First, such

hybrids of E vesca with the octoploids are difficult to make (Yarnell, 1 93 1; Mangelsdorf and










East, 1927) and even more difficult to restore to adequate fertility, and secondly the inheritance

of the trait in the octoploids seems to be different than in Alpine strawberries (Brown and

Wareing, 1965; Clark, 1937). More likely, accidental cross-pollination resulted in seedlings of

pure F. xanana~ssa pedigree, among which was selected the original everbearer. Some, such as

'Louis Gauthier,' a double-cropping French cultivar, may have been simply SD genotypes with

unusually permissive photoperiod or chilling requirements (Fletcher, 1917). Richardson (1914)

suggests that the first true European octoploid everbearer was 'Gloede's Seedling,' introduced in

1866, and this may be the source for most or all European everbearers that followed (Ahmadi et

al., 1990). 'Mabille' and 'l'Inepuisable,' considered by some (Fletcher, 1917) to be pure Alpine

strawberry, followed this, but none were commercially successful. A French priest, Abbe

Thivolet, was among the first to breed for this trait, and after more than a decade of breeding

both octoploids and diploids (Fletcher, 1917) introduced a series of everbearers, culminating in

' St. Joseph', which Darrow (1966) refers to as the "first true large-fruited everbearer." Although

a considerable improvement over the preceding everbearers, 'St. Joseph' and Thivolet' s later 'St.

Antoine de Padoue' were not especially productive, but spurred the development of later

improved types. While EB plants derived from this source remain in production today, this

source has contributed very little to American breeding.

Although they have been called "long day" cultivars, these varieties lack the characteristic

behaviors associated with true long day plants, in the sense that they do not appear to be

regulated by the length of the dark period, and do not react identically to a short day with a brief

night break of light as they do to long photoperiods (Dennis et al., 1970; Durner et al. 1984), nor

is flowering inhibited by SD (Durner et al., 1984). Rather, these genotypes seem more dependent

on the total amount of light received. Dennis et al. (1970) found no difference in flower









formation in the everbearing cultivar Geneva grown under 12 h photoperiods versus those grown

under a 10 h photoperiod with a 2 h night break, a treatment which often inhibits flowering in SD

plants (Downs and Piringer, 1955; Piringer and Scott, 1964). Marked increases, however, were

seen with 18 and 24 h photoperiods. Additionally, they saw a significant effect of light intensity

when increased from 1200 to 2400 f-c on the 18 and 24 h photoperiod treatments (increases were

also seen on the 12 h and 10 + 2 h treatments, but these were not statistically significant). This

study and the later study by Durner et al. (1984) both noted similar levels of flowering for both

12 h photoperiods and 10 + 2 h night break treatments, suggesting that amount of light, not

duration of either darkness or light, is the critical factor.

"Day-neutrals"

The third distinct flowering habit is day-neutral (DN). Observers of wild American

octoploids had noted that many plants of E virginiana subsp. glauca (then called E. ovalis)

flowered in the summer and fall when most wild and commercial short day cultivars had stopped

(Darrow, 1966). Researchers with the USDA evaluated a large number of E. virginiana subsp.

glauca selections in the 1930s and 1940s, and a many were included in breeding efforts at the

time. A number of everbearing varieties resulted from these efforts, including two, 'Arapahoe'

and 'Ogallala,' (Hildreth and Powers, 1941) that are still in existence today. Whether the trait

expressed in these cultivars was the older EB trait or the DN trait derived from glauca is

unknown, as sources of both are present in the pedigrees, the strong tendency of most remontant

seedlings of 'Arapahoe' and 'Ogallala' to fruit in their first year is distinct from those other EB

parents (Ourecky and Slate, 1967), and more typical of DN genotypes (Ahmadi et al., 1991).

Regardless, the introduction of the DN trait that has had the largest impact came in the 1970's,

when the University of California breeding program, under Royce Bringhurst and Victor Voth,

utilized a single selection ofE. virginiana subsp. glauca from the Wasatch Mountains in Utah to









introduce this habit into commercial strawberries (Bringhurst and Voth, 1984). Strawberries

derived from this source constituted a maj ority of the cultivars planted in California in 1999

(Hancock, 1999).

These were distinct from the everbearing habit in that the plants were truly insensitive to

the length of the photoperiod received, and fruit at nearly the same rate over a broad range of

photoperiods (Durner et al., 1984; Ahmadi et al., 1991). Day-neutral cultivars offered improved

heat tolerance and longer harvest seasons compared with the earlier everbearers, but runner

poorly and are difficult to propagate (Durner et al., 1984). Although day-neutrals appear to differ

from older everbearing types, many researchers have failed to distinguish between them, and in

some cases use the terms interchangeably, resulting in confusion. For the purposes of this work,

"day-neutral" (DN) will refer to the trait derived from E. virginiana ssp. glauca, while

everbearingg" will refer to that derived from 'Pan-American', 'Gloede's Seedling,' or other older

sources, and the generic "remontant" will be used to refer to cases where both are implied or the

author has not been clear in distinguishing.

Other flowering habits

Arguably a fourth flowering habit, poorly characterized at this time, is the

amphiphotoperiodic behavior exemplified by the E. chiloensis selection CHI24-1 (Yanagi et al.

2006). This genotype behaves as a standard SD plant under most conditions, with long

photoperiods inhibiting flowering, but initiates flowers under continuous lighting as well.

Although many will cease flowering altogether (Thompson and Guttridge, 1960), a few

cultivated SD strawberry cultivars may fall into this category, as both Sparkle' (Collins and

Barker, 1964) and 'Sweet Charlie' (unpublished observations) have been observed to flower

under such conditions as well, but thus far little research has been done on this phenomenon.









There has been much interest in recent years in finding novel sources of remontancy, and

workers have identified a number of wild accessions that seem to display varying degrees of

photoperiod-insensitivity or continual flowering, mostly among F. virginiana (Sakin et al., 1997;

Hancock et al., 2001, 2002; Serge and Hancock, 2005a, b). These new sources have not yet been

adequately characterized to determine whether they fall neatly into the DN or EB categories, or

whether they represent truly novel mechanisms. Inheritance studies by Serge and Hancock

(2005a) suggest that there may be multiple genetic mechanisms at work.

Factors Affecting Expression Of Photoperiodic Flowering

Temperature Effects On Flowering

A relationship between temperature and flower initiation was established by early workers,

and was highlighted in the large studies of flowering by Darrow and Waldo (1929, 1930, 1933).

Research has also shown that lower temperatures may permit flowering under longer daylengths

even in normally SD cultivars (Darrow and Waldo, 1934; Darrow, 1936). Hartmann (1947b)

concluded that this was the reason that cultivars grown in the coastal district of California near

Watsonville, where the mean temperature is 170C, were able to flower and fruit through the long

days of summer, while the same cultivars planted inland near Sacramento, where the mean

temperature was 230C, behaved as strict short day plants and produced only a spring crop. In

greenhouse studies he found that plants of four cultivars important to the region all flowered

under long days at 170C, but none flowered at 230C. Three of the four same genotypes flowered

freely under both temperatures under short day conditions, while a fourth, 'Fairfax,' failed to

flower at all under the higher temperature, even under 10 h days. Even everbearing cultivars

under long days are inhibited by high temperatures, as are day-neutral types, though in general

the critical temperature is higher for DN cultivars (Durner et al., 1984; Manakasem and

Goodwin, 2001). Heide (1977) found two of four Scandinavian SD cultivars still flowered under










photoperiods of 14, 16, and even 24 h when grown at 120C or 180C, though flowering was

substantially decreased under the longer photoperiods. The effects were highly dependent on

genotype, however, particularly in the middle of the temperature range, a consistent finding of

such studies. In some cases cultivars that are considered to be short day in some locations may

behave as remontant at other locations 'Climax', a British selection, behaved like a SD plant

when grown in the United States, but as a two-crop variety in England where temperatures are

cooler and summer days are longer (Downs & Piringer, 1955).

Much like SD photoperiods, which are necessary to develop flower buds, but inhibit the

development of flower trusses, low temperatures allow for flower induction under long

photoperiods, but may not be optimal for fruit production, because lower temperatures slow

development of the flower trusses (Darrow, 1966). Le Miere et al., (1996) found optimum

temperatures for truss development to be about 190C in 'Elsanta'. Temperatures that fluctuate

diurnally seem to be more effective in promoting flower development than continuous

temperatures, even at the same average temperature (Hartmann, 1947a). Such thermoperiodic

rhythms have been shown to have similar effects in other species (Seneca, 1974; Alvarenga and

Valio, 1989) and may allow for more robust entrainment of the circadian clock (Salome and

McClung, 2005).

Vernalization Requirements

Temperature also plays a second role in the expression of flowering, by conditioning the

plant for flowering before the season begins. A certain amount of chilling, generally defined as

time between 00C and 70C, may be required to break bud dormancy and proceed with the normal

cycle of development, though the extent of chilling required is highly cultivar-dependent

(Piringer and Scott, 1964; Voth and Bringhurst, 1970; Durner and Poling, 1986; Darnell and

Hancock, 1996). Increased chilling has been shown to correlate with increases in leaf area and









number, petiole length, and runner initiation (Guttridge 1969; Bringhurst et al., 1960; Piringer

and Scott, 1964; Braun and Kender, 1985; Lieten, 1997). Chilling has been shown to promote

both vegetative and reproductive development (Darnell and Hancock, 1996). Durner and Poling

(1987) found chilling to enhance vegetative growth and reduce flower induction while promoting

floral differentiation. Non-chilled plants produce fruit of smaller size (Harmann and Poling,

1997) and lower quality (Bringhurst et al., 1960) than do the identical genotypes with adequate

chilling.

Because of the related decrease in flower induction, however, chilling beyond the required

amount is of no advantage and can even markedly reduce the number of flowers. Piringer and

Scott (1964), for example, saw substantial decreases in flower number on 'Marshall' with

increasing chilling.

Juvenility and Plant Age Effects

Juvenility may also play a role in the expression of photoperiod-sensitivity In general,

young plants devote more resources to vegetative than reproductive growth, and may not flower

at all during this portion of their life cycle. This allows a plant to attain sufficient size to support

fruit and seed development before flowering (Thomas and Vince-Prue, 1984) Ourecky and Slate

(1967), working with the EB trait, noted that when populations were retained for a year and

scored for a second season, more plants were determined to be everbearing (a finding supported

by unpublished worked cited in Scott & Lawrence (1975)), casting a degree of doubt on earlier

work that utilized only first year data. However, Ahmadi et al. (1990) found that DN progeny

generally flowered within four months of germination. While not an issue for plants in

cultivation, this effect may have serious impacts on the ability of breeders to make selections

based on flowering habit. Ito and Saito (1962) found that older plants responded more robustly to










both photoperiod and temperature. This may have been in part a function of plant size, although

Hartmann (1947a) found that a single intact leaf was enough to perceive inductive photoperiods.

Growth Pattern Differences Between Plants Of Differing Flowering Habits

In addition to the differences in flower production, strawberries of differing flowering

habits also differ in terms of plant architecture and growth patterns. The production of runners

varies considerably among SD, DN, and EB genotypes. Runner initiation is affected by

photoperiod and temperature as well. In SD cultivars, there appears to be a balance between

runners and inflorescences ranging from almost no runners and many flowers under optimal SD

conditions, through an intermediate area where flowers and runners coexist, and then solely

runnering under the longest days. SD plants will runner in response to short days with a night

break, and they tend to shift from reproductive to vegetative growth under high temperatures

(Piringer and Scott, 1964; Durner et al., 1984). Interestingly, DN cultivars, while flowering in a

photoperiod insensitive manner, seem to retain a sensitivity to photoperiod in respect to

runnering, with the number of runners increasing under long days, night break, or high

temperature conditions. This is in contrast to EB types, which do not show photoperiodic or

temperature changes in runner initiation (Durner et al., 1984). In many, though not all cases

(Durner et al., 1984) EB genotypes runner very little, which may be a barrier to efficient

propagation.

Nicoll and Galletta (1987) also noted differences in plant architecture between DN, EB,

and SD plants. Under long days, the main axis of SD and weakly DN plants remains vegetative

and most axillary buds develop as runners, while under short days, the main axis terminates in an

inflorescence and an upper axillary bud develops as a branch crown. In EB plants, under all

photoperiods, most or all axillary buds that develop do so as branch crowns, rather than runners,

and it is the termination of these crowns with inflorescences that results in most flowering,










though the main axis will occasionally terminate in a flower and continue growth from a side

shoot as in SD plants. In DN plants, by contrast, the growth is characterized by very low rates of

branch crown formation, with upward growth quickly terminated by a terminal inflorescence and

continued from an upper bud, with reduced development of leaves, runners, and branch crowns.

Interestingly, these growth habits closely parallel those seen in E. vesca by Brown and Wareing

(1965), but in that case were found to associate closely with the runnering locus, or a gene close

by it, rather than the seasonality locus, described below. The runnerless gene from 'Baron

Solemacher' conveyed a growth pattern like that observed in DN plants, while runnerless plants

descended from 'Bush White' had a many-crowned habit like that of EB octoploids, and the wild

type E. vesca was similar to the SD octoploids.

Inheritance Of Flowering Habit In Strawberry

Inheritance of photoperiod insensitivity has been described in a number of species. Most

commonly, it is a single gene trait, conferred by either a dominant allele, as in rice (Oryza sativa

L,) (Chandrartna, 1953), sweet pea (Lathyrus odoratus L.) (Ross and Murfet, 1985), jute

(Corchorus sp.) (Joshua and Thakare, 1986), and tetraploid Sea Island cotton (Gossypium

barbadense) (Lewis and Richmond, 1960), or a recessive allele, as in diploid upland cotton

(Gossypium hirsutum L.) (Lewis and Richmond, 1957), cucumber (Cucumis sativus L.)

(Dellavecchia and Peterson, 1984), or okra (Abelmoschus esculentus (L.) Moench) (Wyatt,

1985). In a few instances, the inheritance has been shown to be a more complicated mechanism,

such as the two dominant alleles at separate loci responsible in some hexaploid wheat (Tritcum

aestivum L.) (Maystrenko and Aliev, 1986) or the three genes believed to be involved in sesame

(Sesamnum indicum L.) (Kotecha et al. 1975).

Brown and Wareing (1965) demonstrated that seasonality in E. vesca is conveyed by a

single gene, which they designated S (referred to as SFL by some later authors (Albani et al.,










2004)), with the perpetual flowering habit displayed by the homozygous recessive. They further

demonstrated that the trait was independently inherited from the non-runnering habit, which

proved to be another recessive trait at a different locus. Federova (1937) may have identified

seasonality in E. vesca as a recessive trait earlier, but it is unclear with which species he was

working. Ahmadi et al. (1990) expanded on this by observing that the California E. vesca, which

differs significantly from the European E. vesca morphologically, appears to have two additional

genes conveying photoperiod sensitivity, yielding a 1:63 ratio in the F2 Of a cross between Alpine

E. vesca and local California plants.

Richardson (1914) conducted some of the first studies of the inheritance of the EB trait.

Using 'St. Antoine de Padoue' and 'Laxton's Perpetual', both derived from 'St. Joseph', as

sources of the trait, he found ratios of 1:1 and 3:1, suggesting a simply inherited character.

A considerably different model was that of Clark (1937), who investigated the

expression of the EB trait from 'Mastodon', an everbearer derived from 'Pan-American', in

breeding populations grown in New Jersey. In general, there was a strong trend towards

producing approximately one third everbearers in crosses of EB with non-everbearers

(presumably SD), with an average of two-thirds in crosses of EB x EB, whereas Mastodon selfed

yielded 80% EB, though in a small population. Clark also cites an earlier EB x EB cross,

reported by Macoun (1924), which yielded slightly less, 56.29% EB. In nearly all cases, crosses

of SD x SD, in which one of the parents had one EB parent, yielded no EB progeny. There were,

however, exceptions. One SD plant with an EB parent, N.J. 220, did give some EB offspring in

one cross. Additionally, two everbearers, N.J. 1 and N.J. 8, produced very low numbers of

everbearers in their progeny in crosses with non-everbearers (0% and 8.8%, respectively) and

when selfed (0% and 11.9%, respectively). Clark concluded that the results suggested a complex,










polygenic inheritance, but with evidence that the character is largely, though not wholly,

conveyed by a maj or, dominant allele, possibly modified by other genes.

Later work attempted to clarify this model, although with limited success. Powers (1954)

performed a partial diallel crossing of three EB and seven SD genotypes and developed a three

locus model with four dominant alleles, A ', A, B, and C. conveying the everbearing trait with

varying strength, ranging from A >A>B>C. Any of these alone was theorized to be inadequate

for expression of the trait, as was aaBC_, but A or A plus a dominant allele at either of the

other loci resulted in the everbearing character. He also theorized that as many as four recessive

genes account for the presence of EB plants in the SD x SD progenies.

Although mostly adequate to explain the observed data (though 5 of his 49 families do

exhibit high chi-square values with this model), Powers' model assumed that all seven of the SD

parents in his study were of the same genotype, aaBbCc. Unless there is a significant unknown

selective value to the heterozygote at the B and C loci, it seems exceedingly unlikely that all

three unrelated short day genotypes would be double heterozygotes.

Two confounding factors may have been at work here. First, the everbearing parents used

are primarily breeding selections, so the source of the trait cannot be readily ascertained. Since

the Cheyenne USDA breeding program was utilizing F. virginiana ssp. glauca in addition to EB

cultivars as parents, it is possible that Powers' observations are the result of a mingling of DN

and EB sources. Secondly, Powers used the production of flowers in July, August, or September

as criteria for EB. In light of the fact that Powers' experiments were conducted in the field at

Cheyenne, Wyoming, it may be that low temperatures allowed flowering to continue even during

the long days of summer. Mid-summer day length in Cheyenne is slightly less than 15 h, whereas

the 1971-2000 mean monthly temperatures for Cheyenne in June, July, August, and September










were 16.4, 19.8, 18.8, and 13.60C, respectively (National Climatic Data Center, 2001). Heide

(1977) found that at 180C, three of four SD cultivars still initiated at least some flowers under 14

h days, and thus Powers may have, in fact, been observing everbearing behavior in non-

everbearing genotypes. Later studies may have been affected similarly.

Ourecky & Slate (1967) examined 46 progenies and suggested complementary dominant

genes segregating in an octoploid manner. This is somewhat at odds with later work suggesting

that octoploid strawberries are diploidized (Arulsekar et al. 1981), despite recent hybrid origin.

More recent work by Sugimoto et al. (2005) found a 1:1 ratio of EB : SD in a cross of 'Ever

Berry' (EB) x 'Toyonaka' (SD), as well as in its reciprocal, whereas 'Ever Berry' selfed gave 3:1

and 'Toyonaka' selfed gave all SD progeny. This is in line with earlier work using 'Ever Berry'

as a parent (Monma et al., 1990; Igrashi et al., 1994), and implies a simple monogenic dominant

inheritance.

Although most authors have considered all sources of the EB trait to be genetically

identical, it may be worth noting that these Japanese studies, as well as Richardson's early

studies (1914), the only studies to show clear monogenic ratios, have used cultivars that probably

carry the everbearing trait derived from the European source (or sources). The studies which

have obtained multigene or confusing inheritance ratios have all used American sources of the

trait: Clark (1937), used 'Mastodon', derived from the 'Pan-American' source; Ourecky and

Slate (1967) used a wide range of cultivars derived from either 'Pan-American' or 'Streamliner'

(a seedling of unknown pedigree); and Powers (1954), as previously mentioned, may have been

using a mix of EB and DN parents. Thus the traits, while similar, may have slightly different

genetic mechanisms, or the American sources possess contain modifying genes not found in the

European cultivars.









Characterization of the inheritance of the day-neutrality trait has been clouded by the fact

that studies have indiscriminately included "everbearers" from both the 'Pan-American' source

and true day-neutrals from the E. virginiana ssp. glauca source. At first, day-neutrality was

thought to be the result of a single dominant gene. Ahmadi et al. (1990) found this in an

inspection of nearly 30,000 progeny of crosses between day-neutral and short day plants over the

course of Hyve years. These results firmly agree with a single-gene hypothesis, showing

significant deviation from the model in only one family during one year. Importantly, unlike

early work on everbearers, there were no seedlings that expressed the trait among progenies from

SD x SD. Although the study clearly deals primarily with the glauca source, it is not clear which

remontant cultivars were used and whether all derive from glauca. However, their results differ

significantly from later studies that did include EB from the 'Pan-American' source, which

suggests that all four of the DN cultivars used are derived from glauca.

Serge and Hancock (2005a) also studied the inheritance of day-neutrality, looking at both

cultivated DN and EB cultivars, and apparent novel sources of remontancy found in wild E.

virginiana. Segregation ratios varied widely, apparently suggesting inheritance more complex

than a single gene model. A similar study by Shaw and Famula (2005) using 45 cultivated

genotypes thought to derive their photoperiod insensitivity from the E. virginiana ssp. glauca

source, though not identified in the paper, found strong evidence for the presence of a maj or

dominant locus for day-neutrality, with putative homozygotes flowering more robustly under LD

conditions than heterozygotes. However, it was noted that even homozygous DN plants were not

wholly true-breeding for the trait, suggesting either the effects of other minor loci or deviations

from diploid inheritance.









The system used for scoring progenies may have had a significant impact on the results of

such studies. A primary difference between Richardson' s (1914) studies and many of those that

followed is that Richardson considered everbearers to be individuals that continued flowering

through October, rather than stopping at the end of the summer. Ahmadi et al. (1990) considered

this a more accurate identification of remontant genotypes, but theirs appears to be the only other

study to have done this, as all others reviewed here continued ratings only into August or mid-

September. Serge and Hancock (2003) evaluated five methods of scoring populations for

remontancy, including flowering within 100 days of germination, flowering before a specific

date in the field, flowering under both long and short days in greenhouse or field, and flowering

on newly formed runners in the field. Scoring by flowering within 100 days of germination was

not a good predictor of remontant flowering in the field, but greenhouse observations, if

conducted over the course of an entire season, were well correlated to field performance.

Molecular Markers For Flowering Habit

The ability to screen seedlings for flowering habit at a very early age would be of great

benefit to breeders, saving the time and resources required to plant out and evaluate seedlings

lacking the desired habit. A number of attempts have been made to develop such a system, but

none has seen wide application in breeding.

Albani et al. (2004) used inter-simple sequence repeat (ISSR) markers to identify three

DNA products associated with seasonal flowering in 1,049 plants of a E vesca ssp. vesca

(SFL/SFL) x E. vesca ssp. semperf lorens (sfl/sfl) BC1 population, and successfully converted

these to sequence characterized amplified region (SCAR) markers. Two of these markers were

linked to the seasonal flowering locus at 1.7 and 3.0 cM, while a third, SCAR2, was mapped to

the same location as SFL. This represents the most tightly associated marker for flowering habit

yet developed in Fragaria, but while this work constitutes an important research tool, the impact









of these markers on practical breeding has been limited because of the differences between this

trait and the DN and EB traits of the cultivated octoploid, as well as the lack of commercial

importance of F. vesca.

Kaczmarska and Hortynski (2002) identified a single randomly amplified polymorphic

DNA (RAPD) marker through bulk segregant analysis of a small Fl population, segregating 1:1

for remontancy; however, no attempt was recorded to discern how closely linked this marker was

with the trait or how reliable it was across other progenies.

The previously cited study by Sugimoto et al. (2005) attempted to identify RAPD

markers linked to the EB trait. Five were identified; however the linkages, ranging from 11.8 to

24.3 cM, were rather weak. These weak linkages, coupled with the difficulties sometimes

encountered when trying to reproduce RAPD markers (Paran and Michelmore, 1993), may limit

the practical use of these markers.

Molecular Control Of Flowering In Arabidopsis

Although the effects of genetic variation on flowering, even allelic variation at a single

locus, could be clearly seen in many species, the underlying system often proved complex,

making it difficult to elucidate the roles of individual genes clearly. Among the first steps

towards such an understanding was made through the development of mutant lines of

Arabidopsis thaliana, (a long day (LD) annual species), displaying aberrant flowering patterns.

Reinholz (1945) noted differences in flowering among irradiated Arabidopsis seedlings, and

Redei (1962) followed up on this work by treating imbibed Arabidopsis seeds with X-rays. He

selected four mutants that exhibited altered timing of flowering, designating them constans (co),

luminidependens (ld), and gigan2tea (gi-1, gi-2). All of these lines flowered significantly later

than the wild type control under long days, although the co plants flowered slightly faster under

short photoperiods.









Subsequent work has shown co to be an important element of a complex network of

regulatory pathways governing flowering which are linked specifically to the perception of

photoperiod. There are three distinct parts to photoperiod perception in Arabidopsis:

photoreceptors that perceive light, an internal oscillator that approximates a 24 h cycle, and the

output path to the meristem identity genes involved in flower initiation (Simpson, 2003). CO

itself lays at the junction between the inputs of light quality and photoperiod on one side, and on

the other side a series of genes, such as FT and SOC1, directly upstream of the meristem identity

genes. A simplified diagram of this network is shown as Figure 2-1.

Components Upstream of CO

The perception of photoperiod begins with photoreceptors. Two groups, the

phytochromes (PhyA, B, D, and E) and the cryptochromes (Cryl and 2), react to red/far-red and

blue light, respectively, to entrain the complex feedback loop of the plant' s central oscillator

(Millar, 2003). The central oscillator, in tumn, produces a number of rhythmic outputs, including

CO expression (Suarez-L6pez et al., 2001). This rhythm regulates the base expression level of

CO; however, CO protein abundance is greatly influenced by a number of factors. Key among

these are the further effects of photoreceptors on transcript and protein stability. PhyB has been

shown to promote the degradation of CO protein under red light early in the day, while in the

evening phyA counteracts the effects of phyB and stabilizes CO protein when activated under

far-red light, as do the cryptochromes under blue (Valverde et al. 2004). Another apparent blue

light receptor, FKF l, has been shown to be required to produce the peak in CO transcript level at

the end of the day required to trigger flowering in Arabidopsis (Imaizumi, et al. 2003). A

member of the same family of proteins as CO, CONSTANS-LIKE9, also appears to repress

expression of CO, though the mechanism is currently unknown (Cheng and Wang, 2005), as

does the very similar CONSTANS-LIKE10 (Cheng and Wang, 2006).









Components Downstream of CO

CONSTANS acts directly on two maj or downstream flowering components, Flowering

Locus T (FT) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) (Samach,

2000). Both ultimately act to trigger a suite of meristem identity genes, (in fact many of the same

genes), leading to flowering, and either may be adequate to trigger flowering (Samach, 2000),

though this is disputed (Yoo et al., 2005). In addition to photoperiodic control through CO,

SOC1 also responds to signals from the other pathways regulating flowering in Arabidopsis, the

autonomous, temperature, and gibberellin pathways (Samach, 2000; Moon et al., 2003), and acts

as an integrator of these independent inputs with the common set of meristem genes downstream.

While FT and SOC1 act largely in parallel in inducing flowering (Moon et al., 2005), FT has

been shown able to activate SOC1 to an extent itself (Yoo et al., 2005). Of the two, FT appears

more strongly induced by the photoperiod pathway, while SOC1 is under greater control of the

autonomous pathway and regulated primarily by the gene Flowering Locus C (FLC) rather than

by CO (Moon et al., 2005). In Arabidopsis, another gene, TWIN SISTER OF FT(TSF), which is

very similar to FT has also been shown to perform similar functions (Yamaguchi et al., 2005)

although there is evidence of temperature effects on both genes' activities, suggesting that TSF is

more active at low temperatures whereas FTis more active at higher temperatures (Blazquez et

al., 2003)

Primary among downstream genes is LEAFY (LFY), which is activated by SOC1 and

triggers a number of genes controlling floral development, including API (Nilsson et al., 1998).

FT, however, bypasses LFY and activates API directly (Nilsson et al., 1998), as well as other

meristem identity genes such as SEPALLATA3 (SEP3) and FRUITFULL (FUL) (Teper-

Bamnolker and Samach, 2005).









Acting as a counterbalance to FT is TERMIINAL FLO WER1 (TFL1). TFL1 acts to

suppress the reproductive transition in the meristem, repressing expression of APl. TFL1 is a

member of the same family of genes as FT and TSF, and Hanzawa et al. (2005) demonstrated

that a change of a single amino acid residue in TFL1 is adequate to shift its function to that of a

promoter like FT, though the resulting protein is a weaker promoter of flowering than FT. TFL1

expression is elevated in CO-overexpressor lines, and may in fact mirror CO's promotion ofFT

expression to an extent (Simon et al., 1996). Loss of function t/71 mutants are early flowering

and the inflorescences, unlike the indeterminate growth pattern of wild type Arabidopsis, end in

a terminal flower which is often characterized by abnormal floral organs (Bradley, 1997).

The CONSTANS-LIKE Gene Family

CO is the best understood of a large family of genes, all of which share some basic

characteristics. Specifically, all encode proteins which possess a highly conserved region of 129

amino acids called the CCT ("CO, COL, and TOC", after the three classes of genes that possess

it) domain near the C-terminal end, as well as two zinc-finger type domains side by side at the

other end, though the exact amino acid composition of these domains varies. There are also four

small internal domains present in the canonical CO protein that are retained to some extent in

various other members of the COL family. All are transcription factors, though their exact mode

of action is still unknown.

Griffiths et al., (2003) characterized this family in Arabidopsis, rice, and barley, and

found that three general groups were discernable in all three species, along with a fourth in the

two monocot species. They were able to further subdivide this first group into three subgroups.

The first of these, Group la, contains CO itself and a few closely related genes, specifically

COLl and COL2 in the case of Arabidopsis. These Arabidopsis genes have been shown to be

unable to complement co mutants, and thus are not functionally interchangeable with CO, but do









have effects on circadian rhymicity of leaf movements and expression of cab2 transcript (Ledger

et al., 2001). Many species, including Populus deltoides and P. trichocarpa (Yuceer, 2002) and

Bra~ssica nigra (Lagercrantz et al., 2002), seem to possess multiple members of this subgroup,

but it is not known whether these other members are functionally equivalent to CO. This study

represents the first published documentation of Group la COL genes within the Rosaceae.

Group Ib COL genes are known only in monocots (Griffiths et al., 2003), but Group Ic

genes, represented by COL3 -15 in Arabidopsis, are represented in nearly all species investigated

(Jeong et al., 1999; Griffiths et al., 2003; Hecht et al., 2005). They differ from the Group la

genes primarily in the middle region of the gene, where they lack the characteristic M2 and M3

domains (Griffiths et al., 2003). The role of these genes is poorly understood, but at least one,

COL3, has been shown to have effects on photomorphogenesis, flowering, and inflorescence

development (Datta et al. 2006). Zobell et al. (2005) found members of this family to be the only

COL genes present in the primitive plant Physcomitrella patens, suggesting that they may be the

most ancient representatives of the family. Two Group Ic genes from apple, possibly paralogs

resulting from alloploidy in the species' distant past, represent the only COL proteins previously

characterized in the Rosaceae (Jeong et al., 1999). Unfortunately, the authors did not take into

account the diurnal expression pattern of many COL genes when documenting the expression of

the genes, so it is difficult to derive much meaningful expression information from their work.

Another Rosaceae Group Ic gene, designated PrpCo, was mapped in a peach-almond mapping

population (Silva et al., 2005), but did not correspond to any known quantitative trait loci (QTLs)

for flowering time in almond and was not further characterized.

Group Ib, Id, le, and IV COL genes appear to occur only monocots, but Group II and III

genes are present in Arabidopsis and other dicots. The functions of these groups of genes are still










imperfectly known. Very little information on Group II genes appears to be available. A group

III gene, AtCOL9, has been shown to play a role in flowering, apparently through the suppression

of CO, and, as a result, FT (Cheng & Wang, 2005). Early work presented by the same authors

also showed a related gene, AtCOL10, seems to have similar effects(Cheng & Wang, 2006), and

both appear to be expressed in a circadian fashion. A Group III gene from perennial ryegrass,

LpCOL1, has also been shown to be under oscillator control and is implicated in vernalization

(Ciannamea et al., 2006).

Components of the Flowering Pathways are Conserved Among Species

In some respects, Arabidopsis, a long-day annual, might seem a poor model for flowering

in strawberry, a normally short-day perennial in an entirely different taxonomic family.

However, while there are no doubt critical differences in the physiology of these two species,

mounting evidence suggests that the pathways regulating flowering are remarkably well

conserved between taxa, even those separated from Arabidopsis by considerable evolutionary

distance. The components of the photoperiod and floral pathways in rice, a short day monocot

species, have proven nearly identical to and interchangeable with those of Arabidopsis (Izawa et

al., 2003).

CO has been identified in a number of other species. Besides the model species of

Arabidopsis and rice, a number of studies have suggested that homologs of CO fulfill

comparable roles in other species. These include the dicots Bra~ssica nigra L. (Robert et al.,

1998), Japanese morning glory (Pharbitis nil (L.) Choisy) (Liu et al., 2001), and potato (Solan2um

tuberosum L.) (Martinez-Garcia et al., 2002; Beketova et al., 2006) as well as monocots such as

rice (Yano et al., 2000), common wheat (Nemoto et al., 2003), perennial ryegrass (Lolium

perenne L.) (Armstead et al., 2005; Martin et al., 2005), and barley (Hordeum vulgare L.)

(Griffiths et al., 2003). In some, although the behavior of CO itself appears to be the same,










completely different photoperiod-dependent processes are being regulated. In potato, tuber

development and other associated morphological changes rather than flowering (Martinez-Garcia

et al., 2002; Rodriguez-Falcon et al., 2006). Although modern potato cultivars have been selected

for relative insensitivity to day length, some wild species such as Solan2um demissum and S.

tuberosum ssp. andigena are strictly dependent on short days for tuber formation (Ewing and

Struik, 1992). whereas in aspen (Populus trichocarpa) CO regulates both flowering and the

processes of bud set and growth cessation in response to shortening days in the fall (Boihlenius et

al., 2006). Expressed homologs of CO are also known in species with no known

photoperiodicity, such as apple (for example, GenBank accession EBl141172 and others) and

tomato (Drobyazina and Khavkin, 2006), though their function, if any, remains unclear.

Not only is CO well conserved among species, but so are most of the other components

of the system regulating flowering, both upstream and downstream. Even rice, a short-day

monocot, contains homologs of nearly all the maj or components described in Arabidopsis (Izawa

et al. 2003). Similar pictures are emerging in poplar (Boihlenius et al., 2006; Hsu et al., 2006;

Mohamed, 2006), potato (Rodriguez-Falcon et al., 2006), and apple (Wada et al., 2002; Kotada

and Wada, 2005; Kotada et al., 2006) and this seems likely to be the case in most plant species.

Although the molecular basis for the variation in flowering habit seen in Fragaria is still

unknown, such variations in other crops have been shown to related directly to alterations in this

small group of critical genes. Researchers in Japan have demonstrated that two maj or QTL

affecting heading date, Hd1 and Hd3a, correspond to orthologs of CO and FT, respectively

(Yano et al., 2000; Kojima et al. 2002), whereas a third heading-date QTL, Hd9, has been found

to be tightly linked to the rice homolog of SOC1 (Tadege et al., 2003). Photoperiod insensitivity

in barley has been shown to result from disruptions in the circadian oscillation of CO caused by









mutations in Ppd-H1, a member of the pseudo-response regulator class of genes involved in

circadian clock function (Turner et al., 2005)

Using Model Systems To Understand Development In Rosaceae

Research to date has provided a detailed understanding of the gross physiology of

flowering and development in fruit crops such as strawberry, while work in models such as

Arabidopsis and rice have provided most of what is known about the mechanisms at work at a

molecular level. To truly capitalize on these bodies of work it is necessary to find ways of

integrating information from both.

This is already beginning to occur in apple. Studies have identified and characterized

apple orthologs of many of the components known in Arabidopsis, including AFL1 and AFL2

(apparent equivalents of LFY), MdAP1, MdTFL1, MdIFT, and over a dozen MADS-Box

transcription factors involved in reproductive development (Sung et al., 1999; Yao et al., 1999;

Kotoda et al, 2000, 2002; Wada et al., 2002; Kotoda and Wada, 2005). In general, such research

has shown the components in apple to play roles similar to the homologous Arabidopsis genes.

MdAPI and the pair of LEAFY homologs, MdLFY-1 and M~dLFY-2, have been found to promote

flowering in transgenic Arabidopsis, and appear to be involved in the promotion of flowering in

apple as well (Kotoda et al., 2000; Wada et al., 2002). Similarly, MdTFL1 has been shown to

retard the transition to flowering in transgenic Arabidopsis (Kotoda and Wada, 2005) and

suppression of2~dTFL1 through expression of antisense construct of the gene has been

demonstrated to shorten the vegetative juvenile phase of 'Orin' apple trees from more than six

years to as few as eight months (Kotoda et al., 2006). Some studies, however, have shown that

mechanisms in apple behave differently than would be predicted by the Arabidopsis model. For

example, overexpression of AFL1 in apple, unlike overexpression of the similar LFY in

Arabidopsis, does not result in precocious flowering (N. Kotoda, unpublished research cited in









Kotoda et al., 2006). Regulation of LFY in Arabidopsis is primarily controlled by photoperiod

(Samach, 2000) and apple, an apparently day-neutral species, may regulate flowering through a

pathway not primarily dependent on LFY orthologs.

Classical physiology has already offered numerous hints of what may be occurring in

strawberry at the molecular level. Several researchers (Vince-Prue and Guttridge, 1973;

Thompson and Guttridge, 1960) have suggested that the evidence supports the existence of a

phloem-mobile inhibitor of flowering, synthesized in the leaves. This is reminiscent of the

inhibitory effects of CO in rice (Yano et al., 2002), on FT, which along with TFL1, has been

shown to move from the leaves to the meristem in Arabidopsis (An et al., 2004). Similarly, the

effects of light quality on flowering as shown by Vince-Prue and Guttridge (1973) suggest the

possibility of phytochrome-mediated regulation of a hypothetical CO homolog. To truly

understand what is occurring within the plant in each of these cases, however, will require the

development of the needed molecular tools.

The well-documented complex network of factors affecting flowering in strawberry:

photoperiod, light quality, temperature, vernalization, all have corresponding mechanisms

documented in the molecular models. These mechanisms may not prove identical in strawberry,

and in some cases may prove radically different, yet this knowledge can act as a foundation for

investigations into the interactions between genes and environment that control vital aspects of

the crop's development.

To date little such work has been conducted in strawberry. Yet in many ways strawberry

represents an attractive target for such studies--it has clearly delineated flowering phenotypes, a

short life cycle, ease of hybridization, and convenient plant size. The octoploid genome number

and alloploid nature of the cultivated species represent challenges, but the many diploid relatives









have much simpler and smaller genomes. Like apple, strawberry is an important crop species,

and a better understanding of the genes critical to its development might have economic impacts.

Such knowledge might lead to improved cultivars and more efficient cultural practices,

benefiting the farmer, consumer, and environment.











LIGHT



Photo rece pto rs


Te mpe ratu re


Autonomous






of


post-translational stability
and localization


Circadian oscillator CONSTANS
transcription (CO) s

TFL 1 ~ FT -



Meristem Identity Genes


FY


F LOWVERIN G
Figure 2-1. Simplified diagram showing interactions of genes and environmental factors
governing flowering in Arabidopsis thalianaiiii~~~~~~iiiiii (arrows represent promotive effects,
bars represent repressive effects).


Gibberellin









CHAPTER 3
CHARACTERIZATION OF CONSTANS-LIKE GENES IN Fragaria
Introduction

The initiation of flowering is a critical developmental milestone in most plant species and

represents a vital process for both natural evolution as well as crop production and development.

The mechanisms involved in switching between vegetative and reproductive development have

been shown in model species to be precisely regulated by a number of pathways, governed by

both internal and environmental factors. Among these is photoperiod, which is critical to the

growth and development of many crop species, including the cultivated strawberry. As in many

other species, the timing of flowering in strawberry is an important factor with major impacts not

only on the adaptation of cultivars to particular locales or cultural methods, but also, because of

the relationship with the timing of the subsequent harvest, plays a vital role in determining the

market value of the crop as well.

Despite a narrow genetic base (Sjulin & Dale, 1987), commercial strawberries display a

wide spectrum of sensitivities to photoperiod, ranging in phenotype from strictly short-day (SD)

and "long-day" everbearing (EB) cultivars to day-neutral (DN) types that flower regardless of

photoperiod. Because these different types share overwhelmingly similar genetic backgrounds

and may even co-exist in the progeny from a single cross (Ahmadi et al., 1991), the differences

are likely attributable to the qualitative and quantitative attributes of a small number of critical

genes, thus strawberry provides a unique model system to investigate this important pathway.

The ability to characterize the molecular basis for these differing phenotypes could have

important implications for cultural techniques and cultivar development not only in Fragaria,

but also potentially in the other cultivated members of the Rosaceae, for which timing of

flowering is also a critical factor.









Studies in strawberry allow direct translation of molecular paradigms devised in long-day

and short-day model systems to a single species with ranging photoperiodic flowering habits.

Studies in Arabidopsis have shown that the gene CONSTANS (CO) plays a critical central role in

the regulatory pathway responsible for photoperiodic flowering (Simpson and Dean, 2002;

Corbesier and Coupland, 2005), acting as a dynamic link between photoreceptors, the circadian

oscillator, and the genes governing meristem-identity. Mutations in the CO gene cause delayed

flowering in Arabidopsis, a LD plant (Putterill et al., 1995), and early flowering in rice, a SD

plant (Yano et al. 2000). Its function as a regulator of flowering is well-conserved across species,

having been characterized in both other eudicots (Robert et al., 1998; Liu et al., 2001) and

monocots (Yano et al., 2000; Martin et al., 2004; Nemoto et al., 2003), though the downstream

effect varies between species, either suppressing or promoting flowering. CO is one member of a

large family of CONSTANS-like genes, numbering 17 in Arabidopsis, 16 in rice (Oryza sativa) ,

and at least nine in barley (Hordeum vulgare) (Griffiths et al. 2003). No accounting of these

genes appears to have been previously published in strawberry.

The high degree of conservation in both structure and function, even between monocots

and eudicots, suggests that COL genes likely have important roles in controlling flowering time

across species. A similar situation is likely the case within Fragaria, yet its diverse flowering

habits suggest that regulation may be imparted through novel alleles of known genes,

uncharacterized patterns of expression, or pathways that circumvent photoperiodic influence by

acting on elements downstream from the photoperiodic regulators. This study proposes to

identify, catalog and characterize probable COL genes in Fragaria, describe tissue specific,

temporal and photophysiological expression patterns. The results of this study provide additional









information about into the mechanisms that regulate photoperiodic development. These results

have implications for the evolution of the gene family in Rosaceae.

Nomenclature

Because of the difficulties in coordinating the numbering of family members across

species that may possess different numbers of such genes, a decision was made to simply number

the COL genes in the order they were found, rather than attempting to match numbering of

Arabidopsis genes. In this report, FrCOL is used to refer to genes at a particular locus across

members of the genus Fragaria, whereas FraCOL, FrvCOL, FrnCOL, and FriCOL are used to

designate genes of E xanana~ssa, E. vesca, E. nubicola, and E. iinumae, respectively. Aside from

numbering, genes were named for their apparent Arabidopsis homologs.

Results

Cloning And Identification Of Four CONSTANS-LIKE Genes Of Fragania

A series of EST libraries have been generated by this laboratory, representing transcripts

accumulating in mature xanana~ssa plants (Folta et al., 2005), developing flower buds, various

root tissues, 12 fruit stages (K. Folta et al., unpublished) and E. vesca seedlings (J. Slovin et al.,

unpublished). These EST collections served as a basis for gene discovery based on direct

sequencing, colony hybridization and PCR with degenerate primers.

Simple homology comparisons to sequences within public databases identified

convincing homologs of at least Hyve distinct Constans-like (COL) genes. Four were designated

FrCOL1, FrCOL2, FrCOL3, and FrCOL4, whereas the fifth, which shared the greatest

homology with AtCO and the rice Hd1, was presumed to be the Fragaria ortholog of CO and

designated FrCO. These genes represent the canonical three distinct subgroups of the COL

family as outlined by Griffiths et al. (2003) (Figure 3-1). Close matches to each of these genes

were found among publicly available ESTs in other Rosaceae species as well (Table 3-1).









A full-length cDNA clone ofFraCO was obtained from a flower tissue library derived

from E. xanana~ssa 'Strawberry Festival'. Seven individual clones were identified and

sequenced, and all contained transcripts completely identical except that two contained a

somewhat truncated version of the 3' UTR. The 1,197 bp coding region shows considerable

similarity to AtCO and other previously characterized homologs, with the most similar known

transcript being PdCO1, from Populus (Table 3-1). Comparison of the amino acid sequence to

those proteins designated as Group I suggested that FrCO belongs in Group la (Figure 3-2), a

division that includes all genes experimentally demonstrated to complement the Arabidopsis co

mutant and those believed most likely to play an important role in flowering behavior. The

predicted protein contains a CCT domain and two intact B-Box regions, more closely resembling

similar proteins in other eudicots, rather than the less conserved second B-Box present in some

monocots (Martin et al., 2004). All four middle domains common to Group la COL proteins

were also identified.

Full-length coding sequences for three other FrCOL genes identified could be deduced

from overlapping E. vesca ESTs, as well as part of a fourth. Although these genes contained the

basic structures of COL genes, they bore less similarity to AtCO than did FrCO and appear

unlikely to be the Fragaria ortholog of CO. These were designated FrCOL1, FrCOL2, FrCOL3,

and FrCOL4.

FrCOL1 and FrCOL2 both encode predicted proteins consistent with Group Ic COL

genes, with the maj or difference from the Group la genes being the presence of the M2c (Zobell

et al., 2005) rather than the M2 domain (Figure 3-3). Several copies of both were identified

among E. vesca EST sequences, as well as a single, slightly different, E. xanana~ssa 'Strawberry

Festival' FrCOL1 sequence. FrCOL1 is roughly equal in similarity to both AtCOL3 and AtCOL4









at the amino acid level (55%), with AtCOL3 having slightly higher similarity in the maj or

functional domains. It is most similar to M~dCOL1 and M~dCOL2 from apple, the only previously

characterized COL genes in the Rosaceae (Jeong et al. 1999), and to PrpCO, a COL gene from

peach mapped by Silva et al. (2005). FrCOL2, while retaining a similar structure at the amino

acid level, was not closely related to FrCOL1 at the nucleotide level, suggesting the two genes

diverged some time ago. Among Arabidopsis COL genes, the predicted protein most closely

resembled that of AtCOL5. Based on their relative frequency in BLAST-X searches of Rosaceae

ESTs, FrCOL1 and its orthologs are possibly among the most commonly expressed COL genes

in the family, whereas FrCOL2 and its homologs are expressed at a somewhat lesser level (data

not shown).

FrCOL3 was the least CO-like of the transcripts identified, with a structure consistent

with a classification in Group II. FrCOL3 was found only in F. vesca libraries derived from

young seedlings, and although matches were found among Rosaceae ESTs (primarily in an apple

seedling library), it appears to be a much rarer transcript than the FrCOL1 and FrCOL2 genes.

Only a partial sequence for FrvCOL4 was found among the sequenced ESTs, comprised

of the B-Box region and much of the 5' UTR. Although the full sequence was not available, this

clearly represents a gene distinct from those previously described. The closest match in

Arabidopsis was AtCOL10, indicating this is a likely member of Group III, and if so, all four

groups present in Arabidopsis have also been identified in Fragaria (Figure 3-2). No members of

Ib, Id, le, or IV were found, none of which have thus far been identified in eudicots (Griffiths et

al., 2003).

FrCO/FrCOL Gene Structure

Group I and II COL genes in Arabidopsis possess a single intron located about a third of

the length from the 3' end of the coding region, between the M3 and M4 regions (Fig. 3-4).










Using internal primers designed to include ~400 bp of the coding region and to flank the putative

intron site (Table 3-2), based on the previously identified cDNA, partial genomic versions of

FrCO, FrCOL1, and FrCOL2 were amplified and sequenced from genomic DNA of E

xanana~ssa 'Strawberry Festival', E. vesca FDP815, and E. nubicola FDP60 1 (Figure 5-4.)

Additionally, a product was also amplified in xanana~ssa flower cDNA, using the same primer

sets.

In all cases, an intron was shown to be present at the predicted site in the amplified

genomic product (Figure 3-4). FrCO intron length ranged from 704 to 746 bp. Introns in F.

nubicola, E vesca, and three nearly identical alleles from E. xanana~ssa (FaCO-35) were highly

similar in both sequence and length (704, 707, and 706 bp, respectively), whereas a second F.

xanana~ssa allele (FraCO-C9) was considerably different, containing a 28 bp insertion and

numerous other polymorphisms, for a total length of 746 bp. Somewhat similar to this allele

were those of E iinumae and E. nubicola (Figure 3-5). Alleles also differed significantly in

sequence at one site in the coding region, between the Ml and M2 domains. This region appears

to be characterized by highly repetitive sequence containing multiple CAA codons, and is poorly

conserved between and even within species (Figure 3-5A,B). FraCO-C9 and E. iinumae were

considerably divergent in this region from the other alleles sequenced (Figure 3-5). FrCOL1 was

shown to contain a 82 bp intron in both E.x anana~ssa and E. vesca, similar in length to the

related AtCOL3 (101 bp), but considerably smaller than that ofAtCOL4 (403 bp). Additionally, a

12-bp insertion was identified in a single 'Strawberry Festival' EST, designated 7C. A primer

landing in this insertion amplified a product in all eight octoploid cultivars tested, but not in any

of the diploids, suggesting that this particular insertion may be unique to the octoploids.









FrvCOL2 contained a 118 bp intron, whereas FrnCOL2 a 119 bp intron, both similar in

length to the 88 bp intron of AtCOL5. The cDNA product for FrCOL1 was the same size as that

predicted from the sequenced transcript. However two product sizes were observed for FrCOL2,

consistent with the presence of an unspliced transcript variant, and a partial EST transcript

containing nearly all of the intron sequence was identified among E. vesca sequence data.

Southern blot analysis of FrCO Copy Number

Southern blot analysis displayed a banding pattern consistent with a single FrCO locus in

E. vesca, as in other diploid organisms, showing two bands in those lanes where the probe

fragment was cut once by the chosen enzyme, and one in those where it was not cut. The

cultivated octoploid, E. xanana~ssa, however, showed multiple bands, frequently as many as six

for enzymes that cut with in the probe region (Figure 3-7).

Mapping Of Fragaria CO And COL Genes In A Diploid Mapping Population

The fragment of FrnCO generated by the RN-FaCO primers was found to lack one of the

two HindlII enzyme restriction sites possessed by the corresponding fragment of FrvCO, and this

was used to generate a CAPS marker for mapping (Figures 3-8A, 3-8B). This marker segregated

in a 14:24: 18 (aa:ab:bb) ratio in a subset of the reference population developed by Sargent et al.

(2004), and was found to map to linkage group VI, with linkages to SSR markers E1VFnl53,

UJDFO25, FAC-005, and EMFv160AD, with LOD values of 15.48, 7.79, 11.15, and 13.71

respectively (Figure 3-8C).

No polymorphisms were found between E. vesca FDP815 and E. nubicola FDP60 1 in the

intron and flanking coding region ofFrCOL1. Two polymorphic restriction sites were identified

in FrCOL2, and mapping was attempted first using EcoRV. Because of suspect segregation

ratios and banding patterns that were difficult to resolve, a decision was made to score the

marker as a dominant marker, but the incongruous segregation ratio persisted, yielding a 40:41










(aa:b_) ratio. The first 24 individuals were also scored using the second enzyme, AflHII, yielding

the same pattern seen among these plants with the first enzyme. The resulting mapping data did

not convincingly place the gene on any of the seven linkage groups or in a strong linkage

relationship with any of the established markers.

Comparison Of Strawberry Genotypes Under Varying Photoperiods

In order to test the expression of FrCO and other genes under various photoperiods, a pair of

growth chamber experiments were performed. The first growth chamber experiment consisted of

a comparison of the SD cultivar Strawberry Festival with the DN cultivar Diamante under 8 and

16 h photoperiods. After several weeks under these treatments, these genotypes reacted as

predicted, with Strawberry Festival' flowering heavily under SD and producing numerous

runners under LD, and 'Diamante' flowering vigorously under both photoperiods. The DN plants

produced runners only under the 16 h photoperiod, and even then only produced a very few

runners. Although observations were made, no flower or runner data were collected in this

experiment.

The second growth chamber experiment, under cooler temperatures, compared

'Diamante' to the SD cultivar Camarosa, and to a selection of F. vesca, Hawaii-4. 'Diamante'

again flowered under both conditions, more vigorously under LD than SD. 'Camarosa' did not

runner and flowered only under SD. Unexpectedly, Hawaii-4, a yellow-fruited diploid selection

presumed to be closely related to F. vesca ssp. semperflorens and previously classified as not

photoperiod-sensitive (Oosumi et al., 2006), behaved in a clearly SD fashion, flowering

profusely under an 8 h photoperiod and runnering primarily under LD (Table 3-4).

The expression pattern ofFraCO in the first study was studied by RNA-gel (northern)

blot and subsequent hybridization to a radio-labeled CO probe. Transcript levels varied both

between plants grown under SD and LD conditions, as well as between SD and DN genotypes










(Figure 3-9). Under LD, transcript level in 'Strawberry Festival' peaks rather weakly before

dawn, whereas under SD a strong peak appears in the early part of the day. Transcript levels in

'Diamante', however, are lower under both conditions, with a broader peak in the afternoon,

later than in 'Strawberry Festival', and almost no transcript present under long days.

One concern was that RNA-gel blot experiments may not give precise measurement of

CO transcripts because of potential cross-hybridization with other family members. To verify the

results, gene-specific primers were developed and used to evaluate steady-state RNA levels in a

similar experiment, this time using semi-quantitative RT-PCR. Similar results were seen when

the growth chamber experiment was repeated with 'Diamante', 'Camarosa', and Hawaii-4

(Figure 3-10) using these methods. All cDNA samples were tested with primers for ubiquitin and

the ubiquitin control was used to balance template amount across samples within each set.

Allele-Specific Expression Patterns Differ Betwen Short-Day And Day-Neutral Genotypes

Although allele-specific primers for the FraCO allele types 35 and C9 amplified products

from genomic DNA of 'Strawberry Festival' and 'Diamante', 33 cycles of RT-PCR showed that

while both alleles are actively expressed in 'Diamante', only the FraCO-35 allele appears to be

significantly expressed in 'Festival' at dawn under short days, although a trace of FraCO-C9 is

visible. A more visible band appeared at 40 cycles, suggesting that trace amounts may still be

expressed, although this may be the effect of buildup from slightly non-specific priming (Figure

3-11). Additionally, though the amount of FraCO-35 and FraCO-C9 product present at 33

cycles in 'Diamante' seems similar, the amount of FraCO-35 product present in 'Strawberry

Festival' appears higher than either allele in 'Diamante'. No differences were noted in the

expression of the 7C allele of FraCOL1, which appeared to be actively and equally expressed in

both cultivars (not shown). As before, template levels were adjusted by equalizing with ubiquitin

controls.










Expression Of Other Genes Under Different Photoperiods

Expression of other COL genes in these samples was also been investigated using RNA

from the preceding experiments to see if their expression correlated in some way with relevant

phenomena. From the first experiment, cDNA was generated from RNA taken at the 5 AM time

point (the point of peak FraCO expression under SD conditions, corresponding with hour 8 in

Figure 3-9) and from the low point of FraCO expression, 5 PM, in the SD and LD 'Strawberry

Festival' and 'Diamante' sample sets. These cDNA samples were used as template for RT- PCR

to compare quantitative differences in expression of the CO and COL genes, as well ubiquitin

and actin as controls (Figure 3-12). The previously noted difference in FraCO level was

observed again, and was accompanied by higher levels of FraCOL2 under short days, but

FraCOL1 appears more highly expressed under long day conditions. No measurable levels of

FraCOL3 transcript were observed under either condition in either cultivar. All transcripts

except FraCOL1 were undetectable at the 5 pm time point under both SD and LD conditions,

coinciding with the low point in FraCO expression seen in the previous experiment, suggesting

that they too may cycle diurnally. Control genes were uniform between treatments, suggesting

that the differences observed in specific targets were reflective of actual variations in transcript

level .

The same process was repeated for the entire diurnal time course ofF. vesca Hawaii-4

from the second photoperiod experiment. In this case, very little expression of FrvCOL1 and

FrvCOL3 were seen. FrCOL2 showed some evidence of circadian cycling, but not the large

difference in amplitude between SD and LD seen with FrCO, and in fact seemed to show

evidence of a 12 hour period of variation (Figure 3-13).









Some FraCOL2 Transcripts Contain An Unspliced Intron

Under some conditions, a second product of higher molecular weight was visible for

FraCOL2. The predicted size of this fragment was consistent with the retention of the 1 18-1 19

bp intron shown in the diploid FrCOL2 genes. Additionally, a partial FrvCOL2 EST, DY674801,

was identified in Genbank with more than 95% of the intron, the sequence ending only a few

bases short of the end. Conditions under which the unspliced transcript was detected varied

between genotypes. In Strawberry Festival', it was present only under LD conditions, at the 5

am time point, whereas in 'Diamante' it was visible only under SD conditions, but at both 5 am

and 5 pm (not shown). In F. vesca, in the second trial, however, it was clearly visible at two

points of the day under short photoperiods (Figure 3-12).

Discussion

The Co Gene Family In Fragaria (Rosaceae)

The results of these studies clearly demonstrate the presence of a family of COL proteins

in Fragaria similar to those previously demonstrated in a number of other species, accounting

for all subgroups demonstrated in Arabidopsis and including a likely homolog of CO.

The five members of the COL family identified in this work cover the full range of COL

diversity described in the Arabidopsis, the only eudicot in which the entire family is known to be

documented, with representatives in all subgroups. Unlike in Arabidopsis, only a single member

oflIa, FrCO, was identified. Unless there is significant deviation at the nucleotide level, the

possibility of other la COL genes in Fragaria seems remote. Both the results of the F. vesca

Southern blot, and the fact that no others were isolated in two hybridization experiments, one

probed with AtCO and the other with FraCO, suggest that other such genes may not exist in

strawberry. No evidence of multiple group la loci was seen among ESTs from other diploid

Rosaceae species, although two distinct but closely-related classes of CO were found in apple.









Multiple group la genes are common, even in diploid species, however, and have been

demonstrated in poplar, tomato (Lycopersicon esculentum Mill.), barley, and oilseed rape

(Bra~ssica napus L.) as well as in Arabidopsis (Fig. 3-1), but have been shown not to be the case

in rice (Yuceer et al., 2002; Ben-Naim et al. 2006; Griffiths et al., 2003; Robert et al., 1998). It is

possible that rather than possessing distinct roles, the non-CO group la genes in these species are

merely the result of duplication, coupled perhaps with at least partial loss of function within the

genome, although AtCOL1 has been shown to shorten the circadian cycle when overexpressed in

wild type plants (Ledger et al. 2001). AtCO and AtCOL1 are very similar, even at the nucleotide

level, but AtCO overexpressors show no change in circadian cycle. However it is possible that

reciprocal mutations have separated functions of the original gene into the two genes, with the

current AtCO retaining the ability regulate flowering and AtCOL1 possessing a role in circadian

regulation.

Although FrCO mapped to the same linkage group in the diploid population as the

seasonal flowering locus (SFL), there was no significant linkage with this locus, as 38cM

separate the two, casting doubt on the idea that non-seasonal F. vesca may be FrCO mutants.

Neither of the two QTLs for date of first flower in the diploid mapping population mapped to the

location of FrCO (Sargent et al., 2006a). This suggests that variation in FrCO is not a significant

source of variation in flowering time in this population, and that unlike in other species, such as

rice (Yano et al., 2002), the lack of seasonality is not the result of mutations in the CO gene.

Homologs of all Fragaria COL genes were found in ESTs from other Rosaceae species,

though in no case were homologs of all Hyve genes found in a single species (Table 3-3). This is

most likely due to the limited number ESTs available from a given species, as COL proteins are










generally relatively low in abundance and only apple begins to approach the large numbers of

ESTs need to reliably locate rare transcripts.

Searches of publicly available ESTs identified group la genes in both Malus and Prunus

species, and it appears that there may be two distinct members of the group in apple. Unlike

strawberry, apple does not appear to be photoperiodic (Carew and Battey, 2005). However,

group la COL genes nonetheless appear to be present in all higher plant species thus far

investigated, including some that lack photoperiodic sensitivity, such as tomato (Lycopersicon

esculentum) (Ben-Naim et al. 2006).

Group Ic, homologs of AtCOL3 -1 5 represented in Fragaria by FrCOL1 and FrCOL2,

appear to be among the most highly expressed COL genes in the Rosaceae, constituting 6 of 19

COL ESTs identified in Fragaria, 13 of 20 in Prunus, and 87 of 106 in Malus, of which a large

maj ority are homologs of FrCOL1. Prior to this study, the only Rosaceous genes yet

characterized were members of this group, a single pair from Malus domestic, M~dCOL1 and

M~dCOL2 (Jeong et al, 1999). A very similar gene designated PrpCO was also mapped to Prunus

linkage group G1 on P. dulcis x P. persica F2 map by Silva et al. (2005), but was not further

characterized.

The group Ic genes may be the most ancient members of the family, being the only COL

genes identified in the primitive plant P. patens (Zobell et al. 2005). As with all non-CO

members of the family, the function of these genes is still unclear. Zobell (2006) found no

discernible phenotype in single mutant knockouts for any of the three members in P. patens.

However, work by Datta et al. (2006) has shown early flowering and changes in lateral

branching and root development in Arabidopsis COL3 knockout mutants, and have suggested a









role as a positive regulator of red light signaling. FrCOL1 and its homologs may have similar

functions in the Rosaceae.

Although two polymorphisms suitable for mapping were detected, mapping FrCOL2 in

the diploid reference population proved difficult, and it was not possible to identify linkages with

any published markers. Scored as a dominant marker, one would anticipate a segregation ratio of

3:1, but in this case the pattern was close to 1:1, skewing towards the E. vesca parent. Some parts

of this map have shown considerable distortion (Sargent et al., 2004a, 2006b), including one

marker, BFACT-010, that segregated 79:5 rather than 3:1 (Sargent et al., 2006b). This distortion

of segregation ratios may be due to the parents chosen as parents of the reference population.

Although Sargent et al. (2004a) produced their population from two selfed Fl E. vesca x F.

nubicola hybrids, previous researchers found Fl plants to be self-incompatible, like the F.

nubicola parent (Evans and Jones 1967) or sterile (Dowrick and Williams, 1959). Although the

mechanics of self-incompatibility have yet to be elucidated in strawberry, it may be that this

system remains functional to an extent, resulting in bias in what pollen may successfully fertilize

the Fl plants. Additionally, although sexually compatible, E. vesca and E. nubicola do not

overlap in their ranges and are distinct on both a morphological and molecular level (Sargent et

al. 2005, 2006b; Potter et al., 2000) suggesting that there has been time for divergence between

these species. Such divergence might result in imperfect pairing of chromosomes, and if large-

scale rearrangements exist between the species, may even be reflected in the formation of

multivalents and gametes with chromosomal abnormalities.

FrCOL3 is the sole representative of group II COL genes identified. While maintaining

the general COL structure, group II genes have only one B-Box and the four conserved middle

regions. Though common to both monocots and dicots, comprising 4 of the 17 Arabidopsis COL










genes and 3 of the 16 in rice, their function remains unknown, and this may represent the first

report of such a gene outside of these two model systems. Apparent homologs of FrCOL3 were

identified among ESTs of only one other Rosaceae species, peach, although a distinct transcript

matching the criteria of a group II COL gene was found among apple ESTs as well.

Mining of EST libraries revealed only one partial transcript of FrvCOL4, consisting of

the 5' end the gene. Although we can only guess at the rest of the gene' s structure, this end is

clearly characteristic of a class of COL gene not otherwise represented by the other FrCOL

genes, group III. The B-Box region of group III genes is distinctive because instead of a pair of

B-Boxes, these genes have a single B-Box followed by a modified zinc-finger region. Although

impossible to observe in an EST, these genes are also distinguished from the others in the family

because of their differing intron structure, with two splice locations in the middle of the gene and

a third within the CCT region, none of which correspond to the intron location in the other

family members (Griffiths et al. 2003). This suggests that the differentiation from the other COL

genes is very ancient.

The function of this class of genes is still imperfectly known, but AtCOL9 has been

shown to play a role in flowering, apparently through the suppression of CO, and, as a result, FT

(Cheng & Wang, 2005). A related gene, AtCOL10, was also shown to have similar role in early

work presented by the same authors (Cheng & Wang, 2006), and both appear to be expressed in

a diurnal fashion.

Evolutionary Relationships Among FrCO Sequences

The relationships between the octoploid FrCO alleles and those of the diploid species

reinforce some aspects of the established views of the origins of the diploid genomes of F.

xanana~ssa, while perhaps casting doubt on others.









One allele class, FraCO-35, bears striking resemblance to that of E vesca. Within the

coding region, only the previously mentioned variable site between the Ml and M2 regions

differs, and in fact this region differs considerably even between two selections ofE. vesca,

Hawaii-4 and FDP815. Even within the intron, the nucleotide sequence is 98% identical between

FraCO-35 and E. vesca (Figure 3-5).

The idea that E. vesca may represent a diploid progenitor of the octoploids is an old one,

suggested first by Ichijima (1926) and reinforced by further work, both cytogenetic (Senanayake

and Bringhurst, 1967) and molecular (Potter et al, 2000; Folta and Davis, 2006; also Chapter 5 of

this dissertation) and this result seems to strengthen this view.

The origin of the other component genomes has never been as clear, though a number of

species have been suggested. The sequence of the other type of octoploid allele, FraCO-C9,

does not seem to clearly implicate any of these, but may indirectly point to two species, F.

nubicola and E. iinumae. E. nubicola has long been discussed as a progenitor of the octoploid

species, and was suggested as a possible source of the A or A' genomes by both Senanayake and

Bringhurst (1967) and Potter et al. (2000). Staudt (1989) had proposed E. nubicola, along with E.

vesca, as the ancestors of the hexaploid species E. moschata, but sequence data analyzed by

Potter et al. (2000) implicates E. orientalis, and Lin and Davis (2000) showed that E. viridis was

the likely cytoplasm donor for E. moschata. The range ofE. nubicola, today confined to a

relatively small area of South Asia, does not overlap the ranges ofE. moschata, E viridis, or E.

vesca. If E nubicola is in fact an ancestor of both E. moschata and the octoploid species, this

may suggest that it or a closely related species once existed across a much broader geographic

area.









E. iinuntae has primarily been implicated in unpublished work by DiMeglio and Davis

(cited in Folta and Davis, 2006). Currently, populations ofE. iinuntae are confined primarily to

the island of Hokkaido and the Kuril Islands. The species appears to be an outlier from the rest of

the genus both at the morphological level (Sargent et al., 2004b), and at the molecular level

(Harrison et al, 1997; Potter et al., 2000, Sargent, 2005). E. iinuntae has proven incapable of

producing hybrids with other diploid species (Bors, 2000; Sargent et al., 2004b) but polyploids

derived from the species do not appear to have been investigated and may in fact be capable of

producing allopolyploid offspring in combination with other species.

No diploid species contained the 28bp insertion in the intron of the FraCO-C9 allele, and

E. iinuntae contained 5bp and 7bp intron deletions not seen in FraCO-C9 or any other allele.

However, an inspection of SNPs within the intron on either side of the 28 bp insertion reveals an

interesting pattern--on the upstream side of the insertion, C9 and E. nubicola share the same

base at 7 of 8 SNP sites, compared to only 1 between C9 and E. iinuntae. Downstream of this

insertion, however, more SNPs match E. iinua~ne, with 9 of 18, compared to 6 matches between

C9 and nubicola and 3 sites where C9 did not match either species. Small microsatellite repeats

in the intron, all downstream of the insertion, also seem to more closely resemble E. iinuntae,

with two of the three identical between the two alleles (though the difference between E.

nubicola and the other two alleles is only one repeat in both cases), and the third SSR containing

five repeats of AT in FraCO-C9, four in E. iinuntae, and three in all other sequenced alleles. The

coding regions of the gene are nearly identical among all alleles, with the exception of the

variable region between the Ml and M2 domains, which matched exactly between FraCO-C9

and E. nubicola. Given the level of polymorphism in this region, with even members of the same










species showing variability, this evidence would seem to make a strong case that at least this

portion of the gene originated in E. nubicola.

The division seemingly indicated by the 28 bp insertion may represent a slightly uneven

crossing over event in the species' past, creating a chimeric gene with an upstream end derived

from E. nubicola or a near relative, and a downstream end derived from E. iinumae or an ancient

related species. If this locus lies in a region characterized by frequent crossover or disruption by

interchanges between genomes, this may explain the failure to express of the FraCO-C9 allele

seen in 'Strawberry Festival', for example because of damage to the promoter. Nemoto et al.

(2003) found that the CO locus in one of the three genome pairs of hexaploid was not expressed,

despite an intact coding region, and traced this to an 63 bp deletion in the promoter upstream

from the gene.

It seems unlikely that these two classes represent the entire octoploid complement of

FraCO allele types. Only FraCO-35-type sequences were identified in sequencing of cDNA

from 'Strawberry Festival', and only two identical FraCO-C9 sequences were identified (by the

size difference caused by the insertion and by failure to cut with Xbal) among 16 clones

amplified from genomic DNA of 'Strawberry Festival'. It is possible that alleles are not being

identified by the PCR-based methods used in this study due to mismatches in primer sequence,

or that the FrCO locus has been eliminated from one or more of the component genomes of the

octoploid.

FrCO Expression

In previously characterized SD plants, such as rice (Yano et al., 2000) and perennial

ryegrass (Martin et al., 2004), CO expression patterns remained about the same as that seen in

Arabidopsis. The differences in flowering behavior come as a result not of differences in CO










expression pattern, but from differences in the output, with CO acting as an inhibitor of FT and

SOC1, and hence flowering, rather than promoting them as in Arabidopsis.

In strawberry, it appears that something quite different is occurring. Under short days,

FrCO transcript levels conform to a robust diurnal cycle, though the peak is shifted so that unlike

under SD in Arabidopsis, it occurs during the day, rather than after dusk. Under long days,

however, there is little FrCO expression at all, though there is a perceptible peak during daylight

hours as well. Based on transcript accumulation alone, this suggests that FrCO, like AtCO, is a

promoter of flowering, and that the lack of flowering under long days does not necessarily derive

from an inhibition of flowering, but instead from a lack of promotion, and possibly inhibition

through other mechanisms. Interestingly, even the DN cultivar, Diamante, displayed this pattern,

suggesting that the critical difference between SD and DN cultivars is not one of CO expression.

If so, then flowering under LD conditions in Diamante must be the result of elements further

down the pathway than FrCO. Expression of FrCO in 'Diamante' was slightly different than the

other cultivars, with a somewhat flatter, wider peak. In light of the evidence that one of the two

FraCO alleles in 'Strawberry Festival' is being expressed at greatly reduced levels, while

'Diamante' expresses both alleles robustly, this broad peak may be the result of two or more

alleles expressed somewhat out of phase with each other, with the overlapping curves giving the

impression of a wider peak.

Some caution is warranted in the interpretation of these results, as this data reflects only

mRNA transcript levels. Analysis of transcript levels does not take into consideration the

mechanisms that regulate post-translational regulatory mechanisms. Elegant studies by Valverde

et al. (2004) demonstrate that light-quality driven alterations in CO stability and localization are

critical components of CO action.









The expression pattern seen in strawberry does not appear to have been previously

described, and may represent a fundamental change in the perceived function of CO. In

Arabidopsis and rice, according to the external coincidence model, flowers are initiated or

inhibited in response to whether the peak of CO occurs in daylight or darkness. Without knowing

the dynamics of protein level, it is difficult to say for certain what the mechanism is here, but it

appears that the critical factor is not one of when the peak in CO occurs, but how high it is.

Whether this trend seen at the transcript level is continued at the protein level, where further

regulation may occur (Valverde et al., 2004), remains to be seen. If it is, then some factor other

than the integration of photoperiod and circadian cycle provided by CO is likely responsible for

the perception of photoperiod in strawberry, at least with respect to flowering.

CO transcription is regulated by a number of factors, and a switch between SD and LD

flowering in eudicots may be a result of a change in one of these elements, rather than a change

in the functioning of CO itself. The evidence available does not seem to clearly indicate a

mechanism for the reduced FrCO expression seen under LD, but several mechanisms that either

reduce CO transcription or encourage degradation of the transcript have been identified. A

probable blue-light photoreceptor, FKF l, appears to regulate CO transcript level by repressing

CDF l, a repressor of CO transcription (Imaizumi et al., 2003). Other possibilities include the

Group III COL proteins, as both AtCOL9 (Cheng and Wang, 2005) and AtCOL10 (Cheng and

Wang, 2006) have been shown to decrease CO transcript level in Arabidopsis overexpressing

those genes.

The new evidence also calls into question the role of phytochrome in the regulation of

flowering, and the degree to which it is maintained across species. In Arabidopsis and other LD

plants, far red-enriched extensions of day length at the end of the day accelerate flowering,









whereas red light extensions delay flowering (Kadman-Zahavi and Ephrat, 1974). This has been

explained by Valverde et al. (2004), who found that phytochromes mediated the stability of the

CO protein, with phyB, activated under red light, encouraging the degradation of the protein in

the morning, and phyA, activated under far-red, stabilizing the protein. The phyB receptor also

regulates flowering through the upregulation of PFT1, a repressor of FT (Halliday et al., 2003) as

well as possibly promoting the expression of LFY (Blazquez and Weigel, 1 999).

While this model explains the flowering responses to light quality observed in LD plants, it

becomes clear that something different is happening in SD plants. Vince-Prue and Guttridge

(1973) found that just the opposite occurred in strawberry, with far red extensions inhibiting

flowering if applied at the beginning of the day, and red light extensions inhibiting if applied at

the end of the day. Kadman-Zahavi and Ephrat (1974) grew plants under blue light supplemented

with far red, and found flowering delayed compared to blue light alone or shaded conditions.

While this might seem consistent if FrCO was a repressor of flowering, the expression data seen

in our study would seem to suggest that it is not. In fact, rice, in which CO actually has been

determined to be a repressor of flowering, does not display this pattern, but instead reacts in the

same manner as LD plants (Kadman-Zahavi et al, 1976). Because most of this regulation is

occurring at the level of the protein, it is difficult to gauge what is occurring in strawberry based

solely on our transcript level data.

Kadman-Zahavi and Ephrat (1974) described two clear groups of SD plants, based on this

response. With strawberry, they place Chil pailrl l~lnhean morifolium Ramat. and P. nil, while in the

group of SD plants that respond like LD plants to red and far-red light they place M~imosapudica

L, Amaranthus sp., and Cosmos bipinnatus Cay.. Descriptions from earlier work also suggest

that Salvia occidentalis Sw. is also similar to strawberry (Meij er, 1959), whereas corn (Zea mays










L,), rice, and sorghum (Sorghum vulgare L.) belong to the other group (Kadman-Zahavi et al,

1976; Lane, 1962). Roses, close relatives of strawberries, have been shown to have an increase in

flowering in response to red light extensions at the end of the day (Maas and Bakx, 1995, 1998),

so it appears both groups may exist even within the Roisoideae. If so, this may imply a relatively

simple difference in the mechanisms controlling such responses.

Expression of Other COL Genes

Despite belonging to a common subgroup, FraCOL1 and FraCOL2 displayed rather

different expression profiles. FraCOL2 appears to be expressed in a pattern similar to FraCO, a

pattern previously shown to be common to other COL group Ic genes in Arabidopsis. Microarray

data by Smith et al. (2004) show that AtCOL3 and AtCOL4 follow similar diurnal patterns, with

peaks around dawn under long days. Most other COL genes that have been investigated,

including AtCOL1, AtCOL2, and AtCOL9 (Ledger et al. 2001, Cheng & Wang, 2005) also show

this pattern.

The variation in splicing efficiency of FraCOL2 does not seem to follow a clear pattern,

however it may serve as a means of negative regulation of its function, as the introns in E.

nubicola FDP60 1, E. vesca FDP815, and E. vesca Hawaii 4 all contain multiple stop codons that

would result in a truncated protein. Such a truncated protein would lack the M4 and CCT

domains. Little is known about the functions of these elements. The CCT domain has been

proposed to be involved in the localization of the protein; however the co mutants co-5 and co-7,

with mutations in the CCT domain, localize correctly but do not function properly (Robson et al.,

200 1). Given that the function of FrCOL2 is unknown, it is difficult to speculate on the role of

this truncated protein, if any, but it seems conceivable that it might compete with the functional

version of the protein in its interactions with either DNA or protein.










Splice variants of the CO ortholog PnCO of Japanese morning glory were documented by

Liu et al. (2001), who noted both unspliced and alternatively spliced transcripts in addition to the

correctly spliced version. A maj ority of transcripts contained the full intron, whereas smaller

numbers were properly spliced or retained a small 26 bp segment of intron sequence. The partial

retention of the intron resulted in a frameshift and premature stop codon, but the presence of the

entire intron caused the coding sequence to remain in the correct reading frame. Despite this,

only the properly spliced transcript was able to complement Arabidopsis co mutation in

transgenic plants. The flowering-related gene Proliferating Inflorescence M~eristem (PIM) from

pea, a homolog of the Arabidopsis meristem identity gene AP1, has also been shown to

commonly exist as an unspliced transcript (Taylor et al., 2002).

Transcripts with unspliced introns are moderately common in plants (Ner-Gaon et al.,

2004). Alexandrov et al. (2006) found that about 7% of transcripts demonstrated some sort of

alternative splicing, of which 27% (Alexandrov et al., 2006) to 3 8% (lida et al., 2004) contain

one or more unspliced introns. Ner-Gaon et al. (2004) found that transcripts associated with

physiological flux were more prone to intron retention than those involved in functions such as

metabolism and housekeeping. lida et al. (2004) found that retained introns were more common

in plants that have received a recent stress, such as cold, heat, ultraviolet light, dehydration, and

various hormone treatments. By producing non-functional truncated products or, alternatively,

products that are degraded by nonsense-mediated decay (NMD) caused by frameshifts (Nar-

Gaon et al., 2004), an extra level of regulation in genes that are critically sensitive to changes in

transcript level may be provided.









Unlike FraCO and FraCOL2, FraCOL1 transcript appears to remain at relatively

constant levels through the course of the day. It is the only FraCOL gene expressed in all

cultivars at the 5 pm time point under short days, the apparent low point of the FraCO cycle.

In contrast, the closest Arabidopsis homologs, AtCOL3 and AtCOL4 are both expressed

in a diurnal fashion (Smith et al., 2002), although Jeong et al. (1999) did not investigate diurnal

expression of the apple homologs of the FraCOL1 gene that they characterized, nor is it clear at

what point or points in the diurnal cycle tissue was collected. While both genes were expressed

at the same relative level in all tissues, M~dCOL2 was consistently expressed at a higher levels

than M~dCOL1. And while most tissues showed a uniform but low level of expression, higher

expression of both genes was seen in developing fruit and flower tissue, which may partly

explain the great number identified in apple and peach EST libraries, because many of these

were derived from fruit tissue. In strawberry, however, the maj ority of ESTs are from libraries

derived from developing seedlings not yet producing flowers or fruit, so bias in tissue of origin

cannot entirely explain the preponderance of this group among Rosaceae ESTs in GenBank.

Datta et al. (2006) oberseved decreases in the lateral branching of inflorescences in

Atcol3 mutants, and found evidence that AtCOL3 promotes the formation of branches and

inhibits the growth of the primary shoot during short days. If FraCOL1 has a similar function in

strawberry, it may be that selection for increased numbers of inflorescences or proliferation of

branch crowns has selected for a mutation that results in continual expression of this gene, rather

than diurnal fluctuation.

Similarly, it is also possible that the elevated expression levels for FraCOL1 and the two

Malus genes seen in fruit and flower tissue derives from a role within infloresence development,

and that selection for increased flower number might have encouraged selection for relatively









continuous expression. It would be interesting to examine the expression of this gene in E.

daltoniana, a strawberry species characterized by single flowers (Sargent et al. 2004b). Datta et

al. (2006) demonstrated the necessity of a pair of amino-acids, VP, near the C-terminal, for

binding with COP1 and conferring normal red-light phenotype. FrCO, FrCOL1, and FrCOL2 all

possess this motif, but it is lacking in FrCOL3 (the C-terminus ofFrCOL4 is currently

uncharacterized, although group III genes in rice and Arabidopsis lack this pair), suggesting

possible roles for these first three, at least, in photomorphogenic development.

FraCOL3 was not expressed at perceptible levels in any of the RT-PCR experiments,

though we were able to amplify a weak band from Strawberry Festival' flower cDNA.

FrvCOL3 occurs several times among Fragaria EST sequences, and it is worth noting that all of

these are from developing seedlings, although there are only a relatively small number of ESTs

available from mature plants. In Malus and Prunus, FrCOL3-type transcripts occur several times

in mature and seedling material. Two Arabidopsis orthologs, AtCOL6 and AtCOL7, coincide

with QTLs for rosette leaves at flowering, days to budding, and days to flowering, suggesting

possible roles in flowering, although they share this interval of the chromosome with other

flowering and development related genes such as MADS-Box transcription factors and genes

involved in meristem development and hormone synthesis (Bandaranayake et al., 2004).

Materials and Methods

Plant Material

Six octoploid cultivars of xanana~ssa were used during the course of the work. These

cultivars and the sources from which they were obtained are given in Table 3-5. All were

obtained as rooted plants but were propagated as needed throughout the course of the

experiments. Only the second group of 'Diamante' was used directly from the nursery; all others

were multiple by runners in the growth chamber prior to experiments. E. vesca Hawaii-4 was









obtained as runner plants from the collection of Dr. Thomas Davis at the University of New

Hampshire and propagated by runners in the laboratory for use in the experiments.

Plant Growth Conditions

Plants were maintained on shelves in the laboratory at room temperature (230C), under

cool white fluorescent lighting when not in use in diurnal experiments. The first diurnal

experiment, utilizing Strawberry Festival' and 'Diamante', was conducted in the growth

chamber in enclosed compartments, under either 8 h or 16 h photoperiods under cool white

fluorescent lighting. Each treatment consisted of three 25 cm pots per genotype, containing four

mature plants in ProMix BX soilless medium, and the pots randomized within the compartment.

Temperature was maintained at approximately 230C.

The second diurnal experiment was conducted in growth chambers, with six plants in each

treatment of the octoploid cultivars Earliglow and Diamante, and two each of Camarosa. Ten

plants of E. vesca Hawaii-4 were also grown in each chamber. Plants were grown in 10O-cm

square pots in ProMix BX soilless medium and watered and fertilized as needed. Once again, 8 h

and 16 h photoperiods were used, with a photon flux of approximately 300 Clmol~s *~m-2 at the

level of the soil surface. Photon flux was found to be fairly uniform through the chamber. Three

flats containing two of each E.x a72nanassa cultivar were arranged in the center of the chamber,

and the arrangements of the pots within each flat randomized. E. vesca plants were placed

together in a fourth flat. Temperature was set at 180C day / 160C night. Plants in both

experiments were watered and fertilized as needed. Total number of runners and inflorescences

were counted on each plant at 4, 6, and 8 weeks from the beginning of the light treatments,

except for the E. vesca, for which data was collected only at 6 and 8 weeks.









Cloning of FraCO

A partial EST sequence (CO3 80854) resembling CO was identified among sequences

derived from the octoploid 'Queen Elisa'. A single primer based on this sequence, FaCOtop (5'-

TGGATGTTGGAGTTGTACCAG-3 ') was used along with the M13 reverse primer to amplify a

product from a mixed tissue 'Strawberry Festival' cDNA library by PCR. The product generated

was cloned and sequenced, and a second primer, FaCO-R (5'-

CGGCATTGTTCCTTCATACTAA-3 ) was developed. FaCOtop and FaCO-R were used to

amplify a 344 bp product for use as a probe, using Touchdown PCR as described in Sargent et al.

(2004). This probe was hybridized against approximately 20,000 colonies of a F. x anana~ssa

' Strawberry Festival' flower tissue library in E. coli /Gateway vector.

Identification of COL Genes in Genebank

One Fragaria COL gene, FraCOL1, had previously been identified in the description of

an earlier EST library (Folta et al., 2005). All others were identified by TBLAST-N searches

(Altschul et al., 1997) of Fragaria ESTs in GeneBank, using each Arabidopsis COL gene as the

query, as well as searches using only AtCO CCT domain and B-Box region. Identified Fragaria

sequences were also searched against Malus, Prunus, and Rosa ESTs to identify other Rosaceae

homologs.

Gene Structure Characterization

Primers flanking the full coding region for each of the FraCOL genes were designed, as

well as a primer set (RN) enclosing a roughly 400 bp segment of the coding region believed to

include the putative intron site (Table 3-2). This segment of each gene was amplified from

genomic sequence using Touchdown PCR (as Sargent et al. 2004, except extension time was

increased to 1 min) from 'Strawberry Festival', FDP601, and FDP815, and from 'Strawberry

Festival' flower cDNA. The presence ofintron sequence was verified by comparing the fragment









size generated from genomic sequence to that generated from cDNA and the size predicted from

the EST or ESTs that it was identified from. Combinations of the flanking and RN primers were

used to confirm the absence of other introns.

Southern Blot Analysis

Total DNA was extracted from of E xanana~ssa 'Strawberry Festival' and E. vesca

Hawaii-4 using a modified cold CTAB method (Tombolato et al., in preparation). Ten Clg of each

were digested with four restriction enzymes: EcoRV, HindlII, BamnHI, and EcoRI. Digested

samples were separated by size using gel electrophoresis on 0.8% agarose gels, blotted onto

nylon membranes, and UV cross-linked. A FraCO probe was developed using the RN-FrCO-F

and FrCO-full-R (Table 3-2) with 'Strawberry Festival' genomic DNA in a touchdown PCR

reaction as described above. The probe was labeled by random priming as per manufacturer' s

instructions, hybridized overnight at 600C, and given three washes of 20 minutes each with with

a wash buffer comprised of lx SSC and 0.1% SDS.

Genetic Linkage Mapping COL genes in Fragaria

FrCO was mapped in a diploid reference population developed by Sargent et al. (2004,

2006). This is a F2 population from the cross of E. vesca FDP8 15 x E. nubicola FDP601i, and

consists of 94 individuals. The RN-FrCO primer set (Table 3-2) was used to amplify a product

by PCR from each individuals DNA (30 cycles, 520C annealing, 1 min extension). Ten Cll of

each PCR product was digested in a reaction with 0.3 Cll of HindlII enzyme, 1.3 Cll 10x bovine

serum albumin, 1.3 Cll 10x Promega Buffer B, and 0.3 Cll dH20. Digested product was separated

by gel electrophoresis on 1% agarose gels, stained with ethidium bromide, and photographed

under UV light. FDP601 alleles displayed three bands when digested, whereas FDP815

displayed only two. Size differences allowed the trait to be scored as a co-dominant marker.









Attempts to map the FrCOL2 locus were performed as above, with the following

exceptions: the RN-FrCOL2 primer pair (Table 3-2) was used, extension time was set at 45 s per

cycle, and PCR products were digested with Af71II enzyme. When this showed an unusual

segregation pattern, the process was repeated with the EcoRV enzyme. In both cases, the

FDP815 allele was not cut by the enzyme, whereas the FDP601 allele was. However, due to

incomplete digestion, it was scored as a dominant marker.

Marker data was entered into the JoinMap 3.0 software program and mapped relative to

marker data provided by Dan Sargent, consisting of most of the markers appearing in Sargent et

al., 2004 and Sargent et al., 2006.

Extraction of Nucleic Acids

Except as noted above for use with the Southern blot, DNA was extracted using the

Qiagen DNEasy Plant DNA extraction kit, according to the manufacturer's instructions. Some

genomic DNA was provided by others, namely that of E vesca Hawaii-4, E. iinumae FRA377,

E. bucharica FRA520, and E. mandshurica FME, from Dr. Thomas Davis at the University of

New Hampshire, and E. vesca FDP815 and E. nubicola FDP60 1 and that of the F2 mapping

population from these parents from Dr. Daniel Sargent of East Malling Research.

RNA was extracted using a modification of the pine cone method of Chang et al. (1993) as

described in Folta et al. (2005). Briefly, 1 g of tissue was frozen with liquid nitrogen and ground

with a mortar and pestle, then incubated at 650C for 10 min in a CTAB-based extraction buffer

(2% CTAB, 2% polyvinylpyrrolidone, 100 mM Tris-HCI (pH 8.0), 25 mM EDTA, 2.0 M NaC1,

0.5 g/ml spermidine, and 2.0% beta-mercaptoethanol). Samples were then allowed to cool to

room temperature and equal volumes of chloroform:0ctanol were added and the samples

homogenized using a Polytron T10-3 5 tissue homogenizer at 90% of full speed. The organic and

aqueous phases were separated via centrifugation at 8,000 x g. The supernatant was then









removed, mixed with equal volumes chloroform:0ctanol, and then separated via centrifuge again.

LiCl was added to the resulting supernatant to a concentration of 2.5 M and allowed to

precipitate overnight at 40C, after which it was centrifuged again at 10,000 x g. The pellet was

resuspended in 500 Cll SSTE (1 M NaC1, 0.5% SDS, 10 mM Tris-HCI (pH 8.0), 1 mM EDTA)

and again purified with chloroform:0ctanol, then precipitated with two volumes of 100% ethanol.

The pellet was then washed with 76% ethanol, 0.3M sodium acetate, dried using a SpeedVac,

and resuspended in 50C1l of 10 mM Tris-HCL (ph 8.0), 2.5 mM EDTA. Samples were quantified

by spectrophotometry.

Reverse Transcription and RT- PCR

For the first photoperiod experiment 1 Clg of RNA from each condition was reverse

transcribed using AMV reverse transcriptase (Promega Inc., Madison, WI) as per the

manufacturer' s protocol. The resulting cDNA was diluted 1:10 with TE buffer and used as

template for RT-PCR reactions.

Initial experiments were conducted with the 'Camarosa' SD cDNA set to determine the

likely linear range for each pair of primers. For RN-FrCO, RN-Ubiq, and RN-Actin primers sets,

reactions were conducted at 24, 28, 32, and 36 cycles, and the highest number of cycles which

retained the relative band intensities seen at lower numbers of cycles was selected. RT-PCR

reactions used the RN primer sets described in Table 3-2 using standard 3 step PCR (520C

annealing temperature, 30 seconds extension, for 32 cycles for RN-FrCO and RN-Actin, 28

cycles for RN-Ubiq), separated by electrophoresis on 1% agarose gel, then stained with ethidium

bromide and photographed under UV. Template amounts were adjusted using ubiquitin and actin

RN primer sets (Table 3-2) to assure similar amounts of template DNA between cultivars and

treatments and the PCR repeated and gels photographed (Figure 3-13). Allele-specific RT-PCR

trials used the same PCR conditions, but 33 cycles was used because slightly less product was









anticipated than for with the RN-FrCO primer pair. A second round was conducted at 40 cycles,

with the idea of reach saturation and testing the whether any FrCO-C9 product was present in

' Strawberry Festival'.

The second experiment used a similar procedure, but reverse transcription was done using

ImProm II reverse transcriptase (Promega Inc., Madison, WI) as per manufacturer' s instructions.

The resulting 20 Cll reaction was not diluted but used directly as template in a PCR reaction

identical to above, except that 35 cycles was used for FrCOL2. Template volume used in each

reaction was adjusted using ubiquitin and actin RN primers to equalize template amounts. The

products were again separated by electrophoresis (on a 1.5% agarose gel), stained and

photographed.











Table 3-1. Comparison of amino acid identity (%) of the predicted protein encoded by FraCO to
those of Group la CONSTANS-LIKE genes in apple (Mahts dontestica), castor bean
(Ricinus conanunis), Cottonwood (Popubts deltoides), thale cress (Arabidopsis
thaliana) rape (Bra~ssica napus), Japanese morning glory (Pharbitis nil), rice (Oryza
sativa), wheat (Triticunt aestivunt), and perennial ryegrass (Lolium perenne).




Acc. No. B-Box 1 B-Box 2 M1 M2 M3 M4 CCT Overall
Gene


AY515150
NM121589
AF300700
AY280868
ABO41840
ABO94490
AY553297


MdCO
RcCO
PdCO1
4tCO
PnCO
BnCOal
OsHd1
TaHdl-1

Lpco


z Aalus xdomestica CO gene assembled from ESTs EBl130204, EBl141442, and DR996733
SR'Rcinus conununis CO gene assembled from ESTs EG683156 and EG686997











Table 3-2. RT-PCR primers for strawberry CONSTANS-like genes and controls, sequence
source, Thl, and approximate size in cDNA (as calculated from sequence).


TM

(oC)


Gene


Primers


Sequence


ctgaatcctgtgaagaac age

tggatgttggagttgtaccag
catctgatcagaaccagttca

catctgatcagaacctcg

ctgaggccgaggctgcttcg

ctgtaccttaacaccctggc

gatggcaacatgctgacggac

tgatgccctcgacatgaage

cccggtecactccggtcage
ttccaacgctgtttccaacg

ctcagcctccatgagcacc gc


EST


CONSTANS-like Genes

FrCO RN-FrCO-F

RN-FrCO-R

FrCO-35 RN-FrCO-35-F

FrCO-C9 RN-FrCO-C9-F

FrCOL1 RN-FrCOL1-F

RN-FrCOL 1-R
FrCOL-7C RN-FraCOL7C-F

FrCOL2 RN-FrCOL2-F

RN-FrCOL2-R

FrCOL3 RN-FrCOL3-F

RN-FrCOL3-R


Control Genes

FrActin1 RN-Actin-F

RN-Actin-R

FrUbiql RN-Ubiq-F

RN-Ubiq-R


265

256

403


117

411

(531)'
418


CX661699

DY674862
CO817106

DY669887

DY671835

DY667001

DY673134


DV439971 430


CX661133 414


tggctgtgcacgatgattgc
taacttcccaccagatatcc

aaccaaccgtecaacaatcccaac

acc ggatcagcagaggttgatctt


z Sequence obtained in the course of this study

Y Approximate size in transcripts containing unspliced intron

x 5' portion of transcript sequenced from library using universal primers











Table 3-3. GenBank accession numbers for Fragaria COL genes and other Rosaceae orthologs.


Prunus
persica


Prunus
dulcis


Prunus
armemiaca


Fragaria Arabidopsis
COL gene orthologs


AtCO
FrCO
DY675636
DY67203 5
DY669597
CO380854


Malus sp.



DR996733
CV627730
CO754283
EBl32548
EBl30204
DR996467
EBl41396
EBl48115
EBl41172
EBl29058
CV998001
CV883514
EBl41442
CV657475
EBl41442
CV657475


Rosa sp.


DY637206
BU046876
BU046875


AtCOL3/4 AF052584
(MdCOL 1),
AF052585
(MdCOL2),
and 66 ESTs





AtCOL5 EBl57350
and 20 other
ESTs


DY644780
DY643071
DY641101
DY635684
DY635102
DN555580
DN555698
BU042239
(PrpCO)
BU041471
DY644780


CVO49551
CB821542


EC589436


FrCOL1

CX661699
CX661092
CX661512
CX662230
DY674862
CO817106

FrCOL2
DY669887
DY671835
DY674801


BI977339
BI977724


AtCOL6/16


DN554754
DN553950


BQ641156


FrCOL3
DY667001
DY673134


AtCOL9/10 EBl44446
EBl44014
CN581286


BU044949
DW351298


BQ103878


FrCOL4
DY674124














HyCO1
OsHd1
TaHd1-1
PdCO1
PdCO2 I
FaCO
PnCO
BnCOal
AtCO
AtCOL1
AtCOL2
FvCOL2
AtCOL5
MdCOL1
MdCOL2
FaCOL1 I
AtCOL4
AtCOL3
AtCOL9
AtCOL10
AtCOL11
AtCOL12
AtCOIII
AtCOL14
AtCOL15
AtCOL13
AtCOL16

FvCOL3
AtCOL7
AtCOL8



Figure 3-1. Cladogram of CO-like genes, including all Arabidopsis and full-length
Rosaceae COL genes as well as all those from other species Group la genes
demonstrated to functionally complement Arabidopsis co mutants
(underlined). NJ tree was constructed based on the full-length predicted amino
acid sequence of each gene. Because the full sequence was not available,
FvCOL4 is not included in the analysis, but appears most similar to AtCOL9
and AtCOL10. Strawberry genes are shown in bold. Groups follow the criteria
of Griffiths et al., 2003.


















AtCO MLKQE-------------SNDIGSGENNR-ARPOlDTCRSNATYHDALMCA 46
BnCOal MFKQE-------------SNNIGSEENNTGPRAOCDTCGSITYHDALNCQ 47
PnCO MLKEE SCEVLDLDVTIGSSSGSRSGNKQNWARVCDICRSAAC SVYCRADLAYLCGGCDAR 60
FaCO MLKEE-------------SNGAAAAN--SWARVCDTCRSAPCVCADALSGCDAT 45

Consensus M:K:E S.. : : .R CD C S C:VYC:AD AYLC .CDAT


AtCO VHSANRVASRHKRVRBCESCERAPAAFLCEADDASLCTACDEHAPRHQVL 106
BnCOal VHSANRVASRHKRVRBCESCERAPAAFMCEADDVSLCTACDEHAPRHQVV 107
PnCO VHGANTVAGRHERVLT BCEACESAPATVICKADAASLCAACDDHAPARHV 120
FaCO IHAANRVASRHERVWFCEACERAPAALLCKADAASLCTACDIHAPRHQVL 105

Consensus :H.AN VA.RH:RV BCE:CE APA:.:C:AD .SLC:ACD ::HSANPLARRH:RVP :

M1
AtCO PI SG--------NSFSSMTTTHHQSEKTMTDPEKRLVVDQEEGDAEEVSLP 157
BnCOal PITG--------NSCSSLATANHT---TVTEPEKRVVLVQE---Dg TSLP 149
PnCO PI SGTLYGPPTSNPCRESSMMVGLTGDAAEEDNGFLTQDAEETMEDEASLN 179
FaCO PI SG-------------GQIVVGSTPADTTED-GFLSQEGDEEMEDEASLN 151

Consensus PI:G : : : D OT


AtCO ---NSD- KNNNNQNNG----------------LLFEDEYLNVYSM KF E -- 194
BnCOal ---NSDNHNNNNQNNE----------------LLFQDDYLDAYSMKFQNP 190
PnCO PNPNPHPNPVKSNNSTNMCKGGNNNNNEMSCAVEAI~DAYLAESCNFEKY- 237
FaCO PVKNSHSHNSNNNNNP------NSNNNGFFFGVE-1~DEYLDVYSDNF(T-0

Consensus N.I : :.N.:D YL:L.::.S. : F



AtCO SQHQQNCsPQ-S gcRVPQT4----S'GGDVPKE--SRHCNQNQNKGSGHN 247
BnCOal TQHKQDCTVP EK-- NJGGDRVVP LQL~EE-----TRGNLHHKH--IYSGHN 239
PnCO INQQQNYSVPQRNMS'TRGDSIVP-NIGKNQFHYTQGLQQNH-AFCEWMIT 294
FaCO TNDQHSYGVPHK-I SfGGDSVVPVQTGEGKVTQMQMQQKHNFH--QLGMEYESSKAG 259

Consensus :..VP .' GD :VP:: : : H:


AtCO NG SINHNAY I S SMTGVVM ESTADIVTTASHPRTPKGTVEQQgPDPASQM ITVTQLPE 307
BnCOal NGSINHNAYNPSTDVEA 'DTSHPKTHKGKIEKLPEPLIQIL-----SMR 294
PnCO D-----MVSI SSMOVGVVPE STLBDTSI SHSRASKGTIDLFSGPPIQMPPQLQLQMR 349
FaCO DGSI SHTVSVSSMOVGVVPDSTMfiEMSVCHPRTPKGTIDLFNGLTQP-LSMR 317

Consensus :: .SM:...VP:.* : H: G: Q: :SMR


CCT
AtCO AIRVLRYREKRKTRKFEKTRYASRAYAEiRPRVNGiRFAEEEAEGNTLY- 365
BnCOal ARVLRYREKKKRRKFEKTIRYASRKAYAERRPRINGRFAKIEEEQYTLYD 354
PnCO ARVLRYREKKKTRKFEKTIRYASRKAYAETRPRIKGRFAKEDDEDIYPME 409
FaCO ARVLRYREKKKTRKFEKTIRYASRKAYAEARPRIKGRFAKEDDEDMSSME 377

Consensus ARVLRYREK:K RKFEKTIRYASRKAYAE RPR::GRFAKE : :.V :


AtCO GYGIVPSF---- 373
BnCOal GYGIVPSFYGQK 366
PnCO GYGIVPSF---- 417
FaCO GYGIVPSY---- 385

Consensus GYGIVPS:








Figure 3-2. Alignment of the predicted amino acid sequence for FraCO with those of

AtCO and functionally confirmed dicot homologs, with major conserved domains noted.








































HSANPLSHADERVPVTPFYDSVNSATDSVPAVKSAVNFLNDYSVGEAREA 136
HSANPLSSRHDRVPVTPFYDSVNSAANSVPVVKSVVNFLDDYSVGTSREA 136
HSANPLSRRHERVPVBPFYDSLNSGKSDA----AAVNLLDDYS-G--TEA 126
HSANPLARRHERVPVTPFYDSVSSDGSVK---HTAVNFLDDYSIGSREEA 177
HSANPLSRRHERVPITPFYDAVGPAKSAS----SSVNFVDE---DG--- V 121

HSANPL: .:RVP: PFYD::... : FV:::: DG



ASWLLP-NPPAM------------------ENPDLNSGQ-YFEDMLYGVP 176
ASWLLP-NPPAM------------------ENPDLNSGQ- YFEDLLYGVP 176
ASWLLP-NPP-----------------------DLNSGQ-YFDSLLYGPP 161
ASWLLLPNPPTTTTATAGIVAVTSAEEVPGDSPEMNTGQQYFDPLLYGV- 236
ASWLLA--- ----------------------KEGIEITN--F---DY--P 144

ASWLL ::: :F DLDY

M3
LEDAQEQNsSCTDGVVP EQ KNMQPQLVNDHSFEIDFSAA SPVGHACRVS 236
LEEAQEQNSCHADGVVPV Q KNMQP LLVNDQSFELDFSAG SPVGHACQVS 236
KTEAQEQNSS!.TDGVVPV Q KSAQP-----QSFEMELPG- SPY---LQVS 208
KVESLEQNSSMTDGVVPgJTVP VERVITNENCFEMDFTG GSGTGGNCSSS 295
IEVTSEENSSONDGVVPVQ:4KLFLN----EDYFNFDLSA- SIQGN-FQTT 196

:E:NS.I DGVVP : : F::::.. SK : ::VS:

M4
SSDSIVPDDEdAMTDDSNPYNKSMTSAVES-SHPAVQLSSADRERLYKKNF 295
SDISVVPDGtAVT-----------AAVET-SQPAVQLSS VOVRVRRKRNK 284
SPTDVSIVPDcGEMSD----PYPKSISSAVDQLSHPTVQISSDERLYERNK 264
SSHGVPGSVADVS YP YGG PAT SGADPG TQRAVPL T SAREARVMRYRE KRKNRKF 35 5
RT4DVPLVPESqGVT-----------AEMTNTETPAVQLSPREVLYKKNF 245

.::: VP:..: :V ::..:R ARV:RYREKRKNRKF


EKTIRYASRKAYAETRPRIKGRFAKETEVEIEAEPMCR-----GVS 340
EKTIRYASRKAYAETRPRIKGRFAKETEVEIEAERMCR-----GVS 329
EKTIRYASRKAYAETRPRIKGRFAKETEVEIEAERLCR-----GVS 309
EKTIRYASRKAYAEMRPRIKGRFAKETDTNESNDVVGHGGIFGGVT 406
EKTIRYASRKAYAEMRPRIKGRFAKETDSRENDGGDVG--VYGGVS 294

EKTIRYASRKAYAE RPRIKGRFAK1.T: :G:VP:F




.Alignment of the predicted amino acid sequence for FrvCOL 1 with possible

apple and Arabidopsis homologs, with major conserved domains noted.


B-Box 1 B-Box 2
FCRADSAFLCVNC DSKI HAANKLASRHARVWIC EVC EQAPAHVTC KADDAALCVTC DRD I
FCRADSAFLCVNC DSKI HAANKLASRHPRVWJICEVC EQAPAHVTC KADDAALCVTC DRD I
FC RADSAFLC INC DTK IHAANKLASRHARVWI4CEVC EQAPAHVTC KADDATLCVTC DRE I
YCRPDAAFLCLSC DSKVHAANKLASRHARVWNCEVCEQAPAHVTCKADAAALCVTCDRDI
FC RADAAFLCG DC DGK IHTANKLASRHE RVWI~CEVC EQAPAHVTC KADAAALCVTC DRD I

:CR.D:AFLC .CD K:H:ANKLASRH RVW:CEVCEQAPAHVTCKAD A:LCVTCDR:I


MadCOL1 ------------------------------------------MLCDKSTL 16
MadCOL2 ------------------------------------------MSCDQATL 16
FaCOL1 -------------------------------------------AK3DCSTT 16
AtCOL4 MDP TWI DSL TRSC EANSNTNHKRKRE RE TLKHRE KKKKRFRE RKMASKL CDSC KSATAAL 60O
AtCOL3 -----------------------------------------MASR3DCSAT 18

Consensus:: n***T


MadCOL1
MadCOL2
FaCOL1
AtCOL4
AtCOL3

Consensus



MadCOL1
MadCOL2
FaCOL1
AtCOL4
AtCOL3

Consensus



MadCOL1
MadCOL2
FaCOL1
AtCOL4
AtCOL3

Consensus



MadCOL1
MadCOL2
FaCOL1
AtCOL4
AtCOL3

Consensus



MadCOL1
MadCOL2
FaCOL1
AtCOL4
AtCOL3

Consensus


MadCOL1
MadCOL2
FaCOL1
AtCOL4
AtCOL3

Consensus





Figure 3-3











791 bp 746 bp 362 bp
79 bp 70 bp 36 bp

796 bp 704 bp 362 bp


784 bp 703 bp 362 bp


I~ ~

~iriy~if 310hR


688 bp 119 bp 383 bp


FraCO-C9

FraCO-35

FrvCO

FrnCO

FriCO


799 bp


697 bp


362 bp


FraCOL1- 7C


FrnCOL1

FrvCOL1


FrvCOL2

FrnCOL2


620 bp


82 bp 310 bp


688 bp


118 bp 383 bp


Figure 3-4. Structures of COL alleles from Fragaria species: xanana~ssa Strawberry
Festival' (Fra), E. vesca FDP815 (Fry), E. nubicola FDP 601 (Frn), and F.
iinumae FRA377 (Fri). Thinner black lines indicate introns, dark grey
indicates conserved domains, and crosshatching indicates maj or indel
polymorphism sites.


nl CI
82 bp 322 bp












Fe. vesca


F. x ananassa A (35)


F. x ananassa B (C9)


'i I F. iinumae
F., nubicola
0.0 0.01 0.02 0.03 0.04 0.05




Figure 3-5. NJ phylogram tree of intron sequence divergence among FrCO alleles.













































F, nubicola FDP601



F. xananassa Festival-C9



---. x ananassa F~estival-35



F. vesca Hawani-4



F, vesca FDP81~5



F. bucharica FDP520



F, iinumae FDP377
0.0 0.1 0.2 0.3 0.4


(A)


F. vesca Hawaii-4
F vesca FDP815
F. nubicola FDP601
F x ananassa 'Festival' C9
F x ananassa 'Festival' 35
F. bucluvica FDP520
F. iinumae FRA377


F. vesca Hawaii-4
F vesca FDP815
F. nubicola FDP601
F x ananassa 'Festival' C9
F x ananassa 'Festival' 35
F. bucluvica FDP520
F. iinumae FRA377


CTGTGAA--ACAGC-AATAGCCACAACAGTAACA-ACAACAAATCACG-A
CTGTGAAGAACAGC-AATAGCCACAACAGTAACA-ACAACAAATCACG-A
CTGTGAAGAACAGC-AATA----------------ACCACAAATCACA-A
CTGTGAAGAACAGC-AATA----------------ACCACAAATCACA-A
ATCTGAATCCTGTGAGACAGCAATAACT-------ACAACAAATCACG-A
ATCTGAATCCTGTCAGGACAGCACCACGCACCAG-------TACCAATCC
ATG TGAAC GGATAACAAT TTTCACACAGGAAACAGC TAT TG -- --AC CCATGAT -TAC

AAAAGATCTCTCGGTGAGTGTAGACTGACTGGGGACA

AAC AAC GGATTC TTC TTC GGAGTGGAGGT TGATGAGTAC TTGGAC CTTG TGGAG TACAAC
AAC AAC GGATTC TTC TTT GGAGTGGAGG TTGATGAGTAC TTGGACTCTTG TGGAG TACAAC
AACAACGGATTCTTCTTTGGAGTGGAGGTTGATGAGTACTTGCTTGAAAC
AACAACGGATTCTTCTTCGGAGTGGAGGTTGATGAGTACTTGCTTGAAAC
ATCCACCAACGGATCTCTCGGAGTGAGGTTGATGAGTACTTGC-TGAACC
GGCCAAGCTTGGTACCGAGGCTCCGGATTCCACTAGTAACGCCATTCTAT


( B)


Figure 3-6. Comparisons of hypervariable section of coding region in FrCO. (A) Alignment of
variable region of seven FrCO alleles, (B) NJ phylogram comparing nucleotide
sequence within the variable region.









F. xananassa


F. vesca


3 4


5 6


1 2 3 4 5 6


3


kii


7,


Figure 3-7. Southern blot of genomic DNA from E. xanana~ssa 'Strawberry Festival' and
E. vesca Hawaii 4, cut with six different enzymes and probed with a portion of
FrvCO. Lane 1=XholI, 2=XbalI, 3=HindlII, 4=EcoR V, 5=EcoR I, 6=BamnHI.


12
















136 bp 403 bp 199 b:
nd Ill EcoR I


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15




aa bb ab ab ab bb ab bb aa ab bb bb ab ab h


nd Ill Hind Ill


RN-FaCO F
Hir

FrvCO
354 bp
Hir

FmnCO i
354 bp


Xba I


r


,p


51.6-
54.0
54.9-
56.0

60.5-
61.7-
63.7-


--FAC-005
,EMFv1 60AD
--EM Fnl53
"'EM Fv010

--FrCO
--U DF25
--FAC-012a


154 b
RN-FaCO R


587 bp


72.5-U UFFxa01 EO3
74.1 EMFv160BC


(B)


(C)


~~ ~J 1,1)


Figure 3-8. (A) Map of the portion of the FrCO product amplified for use in mapping, showing the
locations of distinguishing restriction sites and lengths of the resulting fragments. (B) Gel
showing characteristic band patterns after digestion with Hind III for the F. vesca parent
(lane 1, genotype aa), the F. nubicola parent (lane 2, genotype bb), twelve members of the
F2 (lanOS 3-14), and the 564 bp band of the hHind III size standard (lane 15). (C) Partial
map of linkage group VI (after Sargent et al. 2005), showing the predicted location of
FrCO.











Table 3-4. Mean inflorescence and runner production ofE. xanana~ssa and E. vesca genotypes
under LD (16 h) and SD (8 h) photoperiods, at 18/160C day/night temperature.

4 weeks 6 weeks 8 weeks
Genotype Inflorescences Runners Inflorescences Runners Inflorescences Runners

Long Day
Diamante 1.2 0.0 3.2 0.0 3.8 0.2
Camarosa 0.0 0.0 0.0 0.0 0.0 0.0
Earliglow 0.0 2.2 0.0 3.0 0.0 3.8
Hawaii 4 --0.1 2.8 0.2 3.8

Man t~l Day
Diamante 0.4 0.0 0.6 0.0 1.0 0.0
Camarosa 0.0 0.0 0.3 0.0 1.0 0.0
Earliglow 0.0 2.4 0.0 2.8 0.0 3.0
Hawaii 4 --3.0 1.8 3.3 1.8

































I


Short Day (8 h)


Long Day (16 h)


Festival (SD)





Diamante (DN)


E:a :-..





~ig~F~F~T~:
~eb~~~
~Qd:~
~~"',Y
Fi~


:r 'I
.r


Festival (SD)





Diamante (DN)


Figure 3-9. Northern blot of RNA from Strawberry Festival', a SD genotype, and 'Diamante', a
DN genotype, collected every 4 h, showing expression of FraCO under short and
long day conditions.










Shoi~d8_h~L

--;.-.-1 I~


Long Day (16 h L)


Hawaii-4 (SD)

Camarosa (SD)

Diamante (DN)


~ ~I~ ~


Hawaii-4 (SD)

Camarosa (SD)

Diamante (DN)


- C~I~;
J


3am


7am


11am


3pm


7pm


Figure 3-10. RT-PCR assay of expression ofFrCO transcript under short (8 h) and long (16 h)
days in three genotypes: E. vesca Hawaii-4 (SD, diploid), xanana~ssa 'Camarosa'
(SD, octoploid), and xanana~ssa 'Diamante' (DN, octoploid). Dawn occurred at 8
a.m. in both chambers, and dusk was at 4 p.m. in the short day chamber and at
midnight in the long day chamber.


~c;~


"--U ~r


















FraCGO-35


Fra CO- C9


D


33 cycles


40 cycles


Figure 3-11. Expression of 35 and C9 alleles ofFraCO at dawn under SD, using RT-PCR at 33
and 40 cycles, in 'Strawberry Festival' (F) and 'Diamante' (D).












Table 3-5. Sources of plants used in this chapter.


Genotype


Species


Source


Location


Camarosa
Earliglow
Diamante
Strawberry Festival
Hawaii-4


. xanana~ssa
. xanana~ssa
. xanana~ssa
. xanana~ssa
E. vesca


Garden Gate Nursery
Edible Landscaping
Edible Landscaping
GCREC
University of N.H.


Gainesville, FL
Afton, VA
Afton, VA
Wimauma, FL
Durham, NH












Hawaii-4 (SD)


11pm


7am 11am


3pm


3am


7pm


Figure 3-12. RT-PCR assay of FrCOL2 expression in F. vesca Hawaii-4, showing variation in
splicing efficiency at several points under short days.


Short Day (8 h L)









Short Day (8 h L)


as~~ al memn


Long Day (16 h L)
Inr rrII (rr


Hawaii-4 (SD) agg ga
11pm 3am 7am 11am 3pm 7pm 11pm 3am 7am 11am 3pm 7pm

Figure 3-13. Ubiquitin controls after equalization of RT-PCR samples using RN-Ubiql primer
set to standardize template amounts.


Camarosa (SD)

Diamante (DN)









CHAPTER 4
IDENTIFICATION AND CHARACTERIZATION OF FLOWERING-RELATED GENES INT
Fragaria AND OTHER ROSACEAE



Introduction

Although CONSTANS plays a central role in the integration of photoperiod and the

circadian clock into the regulation of flowering, CO itself does not directly control the

reproductive transition. Instead, CO, along with other inputs, regulates a network of proteins

which through a cascade of interactions eventually trigger the activation or repression of a suite

of meristem identity genes that ultimately govern the developmental fate of the meristem.

In Arabidopsis, the two most important regulators of flowering directly downstream from

CO are FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF;

CONSTANS 1 (SOC1) (Samach, 2000). These two proteins act in parallel to promote flowering,

and are affected by different environmental inputs. A third protein, TERM7INAL FLOWER 1

(TFL1) is closely related to FT, but has an opposing role. Rather than promoting a transition to

flowering in the meristem, TFL1 delays it, causing growth to remain vegetative (Kobayashi et

al., 1999). FT and TFL seem to have fairly direct impacts on the meristem, but SOC1 acts

primarily through the activation of protein called LEAFY (LFY) to promote flowering. FT

appears to be under greater photoperiodic control than SOC1 (Moon et al., 2005). Although

promoted by CO, SOC1 is also under regulation by the other pathways regulating flowering:

vernalization, autonomous, and gibberellin, and serves as the point at which these regulatory

networks integrate into the general flowering pathway (Izawa et al., 2003).

In addition to these components, a number of other genes affect flowering as well. These

include components of the circadian clock (Turner et al., 2005) and photoreceptors (Izawa et al.,










2003), which affect CO transcript and protein levels, as well as components of the other floral

pathways.

Mutations in these genes cause significant changes in flowering time and sensitivity to

environmental inputs in Arabidopsis, as well as in crop plants such as rice (Izawa et al., 2003).

Although the genetic basis for variation in flowering habit in strawberry is currently unknown, it

would seem quite possible that differences in the expression of this small suite of genes lie at the

core of it. By identifying and characterizing the orthologous genes in Fragaria, it may be

possible to pinpoint the location of the mutation or mutations involved in the shift from short day

to everbearing or day-neutral flowering. This could in turn be the basis for molecular markers to

assist in breeding, a target for genetic engineering, or even shape cultural practices by clarifying

the interaction of the different physiological mechanisms. Such knowledge may have impacts

even outside of strawberry, particularly for the other important crops within the Rosaceae.

To that end, efforts were made to identify the genes comprising this network in Fragaria,

and the expression of some of these genes was examined in strawberry plants under long and

short day conditions.

Results

Identification of Rosaceae Flowering Gene Homologs

Screening of Fragaria EST libraries developed in this laboratory (Folta et al., 2005, as

well as others as outlined in Chapter 3) revealed a number of sequences related to transcripts

known to be involved in flowering in Arabidopsis, rice, or apple (Tables 4-1 & 4-2). In addition

to Fragaria transcripts, ESTs from other Rosaceae species were also searched for MADS-box

and FT/TFL1i-like genes, in order to gain insight into the nature of these genes and gene families

within the larger taxon, and to help fill in gaps where no strawberry genes are known but may

exi st.









MADS-Box genes

Six strawberry MADS-Box genes were identified in ESTs, adding to the three already

documented in GenBank. Most had fairly clear correspondence with one of the fifteen

documented MADS-Box genes in apple, although not all had clear homologs in Arabidopsis.

These included genes related to the Arabidopsis transcription factors SOC1, SEPALLA TA3,

PISTILLA TA, SHORT VEGETA TIVE PHASE, SHATTERPROOFl/2, and AGAM~OUS-LIKE14,

as well as one transcript not closely related to any Arabidopsis gene but somewhat similar to the

apple MdM~ADS11. These were designated, in order, Fra;SOC1, FrvSEP3, FrvPI, FrvSVP,

FrvM~ADS1, FrvM4~rDS2, and FrvM~ADS3. These genes are listed in Table 1, and their implied

relationship to the other known Rosaceae MADS-Box genes and a selection of Arabidopsis

MADS-Box genes are shown in Figure 1.

Fra;SOC1 was first identified as a partial transcript (GenBank Accession CO817776)

resembling a MADS box transcription factor among a small EST library developed from whole

plant tissues of F. xanana~ssa Strawberry Festival' (Folta et al., 2005). Because the glycerol

stock of this clone was available, we were able to sequence the gene from the other end,

obtaining the entire sequence. The 642 bp coding region encoded a predicted MADS Box

transcription factor, with high similarity to the Arabidopsis SOC1, with 63% identity at the

amino acid level. The closest overall match annotated in the public databases was VvM4ADS8,

again from grapevine (Vitis vinifera L.), to which it shared 68% identity at the amino acid level.

Other than this strawberry gene and the previously identified relative in apple (Valazideh et al.,

2006), no apparent SOC1 homologs were found among Rosaceae ESTs. A second Fragaria

gene, FrvM4rDS3 (GenBank Accession DY675364), also grouped within the SOC clade, but

more closely resembled AGL14 than SOC1.









FT/TFL1 genes

An EST (DY674124) bearing considerable similarity to the floral repressors TFL1 and

A TC was also identified among sequences derived from cold-stressed seedlings of the F. vesca

selection Hawaii-4. This transcript was initially passed over because BLAST-X searches showed

only partial similarity to known genes. Upon closer inspection, it became clear that the transcript

retained 147 bp of intron sequence at a site near the middle of the gene, which resulted in a

frameshift, causing multiple stop codons and corrupting more than half of the predicted protein.

This likely represents only a partial failure to complete splicing, as the apple (DQ535888) and

Arabidopsis (NM_120465) genes each contain three introns. With the exon sites identified by

comparison to these likely homologs, the spliced transcript yielded a coding region of 666 bp and

encoded a protein with 83% identity to ARABIDOPSIS THALIANA CENTRORADIALIS (A TC),

and a lower 63% identity to the Arabidopsis TFL1 (Figure 4-2), although the proportion of

positive matches, 91% and 85%, respectively, were much closer. The most similar annotated

transcript was from Populus, a "Flowering Locus T-like protein" (BAD22601), with which the

protein shared 85% identity. At the nucleotide level, the most similar annotated sequence was

again from Poplar, PnTFL1 (ABl181 184), with an e-value of e-60, and once again this transcript

was significantly more similar than the nearest Rosaceae gene, MdTFL1-1 (AB052994), with an

e-value of 9e-28.

Efforts to amplify a strawberry ortholog of FT from genomic or cDNA using degenerate

primers based on apple and Arabidopsis sequence were unsuccessful, as was colony

hybridization with Arabidopsis FT as a probe, and no likely candidates were identified in any of

the EST libraries. No Fragaria sequences were identified with significant similarity to the other

members of the Arabidopsis family: TSF, MFT (M~OTHER OF FT), and BFT (BROTHER OF

F7), however apple ESTs resembling M~FT and BFT were identified (GenBank Accessions









EBl134193 and EB13 8045, respectively), as well as a partial transcript of aM2FT-like gene from

almond (GenBank accession BU57441 1). Relationships of apple and strawberry amino acid

sequences with members of the FT TFL1 family in Arabidopsis are shown in Figure 4-2.

Other genes thought to be involved in flowering

Also identified were a number of elements of the circadian clock, including apparent

orthologs of several response and pseudo-response regulators, TOC1, FKFl, ZTL, as well as

several genes with roles in photomorphogenesis, including genes similar to HY5 and

hemeoxygenase (Table 1). Attempts to use degenerate primers based on consensus sequence

from apple, pear, and Arabidopsis LEAFY genes to amplify the strawberry ortholog from either

cDNA library or genomic DNA were not successful. However, a fosmid from F. vesca,

containing the genomic sequence of LEAFY was obtained from Dr. Thomas Davis of the

University of New Hampshire.

FrSOC1 Expression

Surprisingly, RT-PCR assays showed FrSOC1 expression did not parallel flowering as

expected. Rather, in 'Camarosa', FrSOC1 generally followed FrCO expression under short days,

but with a lag of several hours, with a peak near the end of the day and tapering off into the

night. Under long days, however, the expression levels in all but 'Diamante' were extremely

high, and remained so through out the course of the day (Figure 4-3). In 'Diamante', the day-

neutral genotype, however, FrSOC1 expression followed similar patterns under both conditions.

FrTFL1 and FrLFYExpression

Because of the unexpected results seen with FrSOC1, RT-PCR assays of FrLFY

expression were also conducted on the short day octoploid cultivar, Camarosa. Although

expression levels were low, an increase to 36 cycles revealed expression of FrLFY (Figure 4-4)

and Fr TFL1 (Figure 4-5) to be the opposite of that anticipated by the Arabidopsis model, just as









with FrSOC1. Under short days, when 'Camarosa' was initiating flowers, neither was detectable

in leaf tissues. Under long days, when growth was primarily vegetative, however, both were

expressed at all time points, though levels were low.

Discussion

Rosaceae MADS-Box Genes

MADS-box genes play critical roles in the a number of plants processes, especially in the

development of fruits and flowers (Becker and Theissen, 2003), and as such are important targets

of study in crop plants. The Arabidopsis MADS-box gene family comprises roughly a hundred

members, though only one group, the so-called MIKC class which contains 39 Arabidopsis

genes, has been functionally characterized to a significant extent (Becker and Theissen, 2003).

Becker and Theissen (2003) break this class into fourteen groups, one of which, TM8, does not

exist in Arabidopsis. The eight Fragaria MADS-box transcription factors identified in this study

along with those identified by previous researchers represent seven of these groups. An eighth

group, APl-like MADS-box factors, is represented in several other species within the family,

including peach (PrpM~ADS6), loquat (EjAPl), almond (PrdM~ADS2), and several genes in apple

(MdAP1, MdM~ADS5, M~dMADS2, M~dMADS12, and M~dMADS12a), but none are yet known in

strawberry.

The six groups not yet attested in any of the Rosaceae are the AGL12, AGL15, TM~8, TT16,

ANR1, and FLC groups. Of these, FLC is the best characterized, playing a critical role in the

regulation of flowering by non-photoperiodic factors, especially vernalization (Boss et al., 2004).

It is worth noting that FLC orthologs have been identified only within the Brassicaceae, and are

not functional within all ecotypes of Arabidopsis, nor have AGL15 genes (Becker and Theissen,

2003). With the exception of the ANR1 group, none of these groups were attested in the legume

species Medicago, soybean, and lotus (Hecht et al., 2005). The absence of AGL12, AGL15, and









ANR1-type genes may be explained by the absence of root-derived Rosaceae ESTs, as these

genes appear to be primarily active in root development (Becker and Theissen, 2003). The

remaining groups are mostly poorly characterized in Arabidopsis.

Among the most numerous Rosaceae MAD S-box genes are those of the SEPALLA TA

group. The SEP group in Arabidopsis consists of four members that interact with other MADS-

box proteins to shape organ identity in the four whorls of flower development (Ditta et al., 2004).

Two such genes were identified in strawberry, Frak4~rDS-RIN (Vrebalov and Giovannoni, 2002),

with greatest similarity to AtSEP1 and AtSEP2, and a F. vesca EST we designated FrvSEP3.

AtSEP1 and AtSEP2 are very similar to each other, and likely represent recent duplications, and

this may account for the seven apple genes in this group, although in the case of apple, these

duplications may also be the result of polyploidization in the evolution of this species'. The bulk

of the Rosaceae SEP genes most closely resemble the SEP1/SEP2 genes of Arabidopsis,

including FraAL4~DS-RIN, peach and almond genes, and all but one of the apple genes in the

group. FrvSEP3 and the pear gene PbM4~rDS2 (GenBank Accession AB265800) most closely

resemble SEP3, whereas M~dMADS4 does not bear clear relation to a specific Arabidopsis

member, though it most closely resembles SEP4. In Arabidopsis, the SEPALLA TA genes appear

to be largely redundant in function, and plants lacking all three produce flowers composed solely

of sepals (Pelaz et al., 2000). Sung et al. (2000) investigated the expression of two such genes

from apple, M~dnif4DS3 and M~dnB4rDS4, and found very different expression patterns, with the

first expressed only in the floral primordium, whereas the second was expressed throughout

floral and early fluit development, particularly in vascular bundles. Yao et al. (1999) found seven

MADS box genes were expressed in distinct patterns during flower and fruit development. It

seems likely that Fragaria genes within this family also play roles in reproductive development,









although the developmental differences between Arabidopsis, apple, and strawberry fruit require

some caution in predicting function based on these other species.

One new strawberry MADS-box gene, designated FrvM~ADS3, shares some similarities

with the SEP subfamily, but appears most closely related to the AGL6 group, which it shares

with the apple gene MdM~ADS11. The function of these genes is poorly understood. In

Arabidopsis, their expression seems to be confined to floral organs (Becker and Theissen, 2003)

There is evidence that OM4rDS1, an AGL6-type gene from orchid, interacts with OM~ADS3, a

factor involved in flower formation, and promotes flowering when expressed ectopically through

the upregulation of SOC1 and FT in Arabidopsis (Hsu et al., 2003). Similar results were achieved

by Tian et al. (2005) in their study of another AGL6-like gene, DIM~ADS18, from bamboo

(Dendrocalamnus latiflorus Munro).

The AP3 and PISTILLATA class genes are class B organ identity genes, and combine with

class A and C genes to specify petals and stamens, respectively (Krizek and Meyerowitz, 1996).

Both have been previously identified in apple (Yao et al., 2001) and rose (Kitahara et al., 2001).

The three apple mutants--'Rae Ime', 'Spencer Seedless', and 'Wellington Bloomless'--which

produce apetalous flowers and parthenocarpic fluit, were found by Yao et al. (2001) to contain

retrotransposon insertions in introns of2~dPl, abolishing expression and suggesting a critical role

for the gene in both fluit and flower development. The strawberry PI gene identified here might

thus be an interesting target for RNAi- or antisense-based suppression of expression.

Parthenocarpy might be a means to limit malformation of finit, which can result from incomplete

pollination, and might also increase fluit size.

The gene designated FrvSVP most closely resembles the SVP group of MADS-Box genes.

This group appears to play a role in the control flowering in Arabidopsis, with both SVP