Group Title: BMC Plant Biology
Title: Identification of flowering genes in strawberry, a perennial SD plant
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 Material Information
Title: Identification of flowering genes in strawberry, a perennial SD plant
Physical Description: Archival
Language: English
Creator: Mouhu, Katriina
Hytonen, Timo
Folta, Kevin
Rantanen, Marja
Paulin, Lars
Auvinen, Petri
Elomaa, Paula
Publisher: BMC Plant Biology
Publication Date: 2009
 Notes
Abstract: BACKGROUND:We are studying the regulation of flowering in perennial plants by using diploid wild strawberry (Fragaria vesca L.) as a model. Wild strawberry is a facultative short-day plant with an obligatory short-day requirement at temperatures above 15°C. At lower temperatures, however, flowering induction occurs irrespective of photoperiod. In addition to short-day genotypes, everbearing forms of wild strawberry are known. In 'Baron Solemacher' recessive alleles of an unknown repressor, SEASONAL FLOWERING LOCUS (SFL), are responsible for continuous flowering habit. Although flower induction has a central effect on the cropping potential, the molecular control of flowering in strawberries has not been studied and the genetic flowering pathways are still poorly understood. The comparison of everbearing and short-day genotypes of wild strawberry could facilitate our understanding of fundamental molecular mechanisms regulating perennial growth cycle in plants.RESULTS:We have searched homologs for 118 Arabidopsis flowering time genes from Fragaria by EST sequencing and bioinformatics analysis and identified 66 gene homologs that by sequence similarity, putatively correspond to genes of all known genetic flowering pathways. The expression analysis of 25 selected genes representing various flowering pathways did not reveal large differences between the everbearing and the short-day genotypes. However, putative floral identity and floral integrator genes AP1 and LFY were co-regulated during early floral development. AP1 mRNA was specifically accumulating in the shoot apices of the everbearing genotype, indicating its usability as a marker for floral initiation. Moreover, we showed that flowering induction in everbearing 'Baron Solemacher' and 'Hawaii-4' was inhibited by short-day and low temperature, in contrast to short-day genotypes.CONCLUSION:We have shown that many central genetic components of the flowering pathways in Arabidopsis can be identified from strawberry. However, novel regulatory mechanisms exist, like SFL that functions as a switch between short-day/low temperature and long-day/high temperature flowering responses between the short-day genotype and the everbearing 'Baron Solemacher'. The identification of putative flowering gene homologs and AP1 as potential marker gene for floral initiation will strongly facilitate the exploration of strawberry flowering pathways.
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Research article

Identification of flowering genes in strawberry, a perennial SD plant
Katriina Mouhut1,2, Timo Hytonen*t1,3, Kevin Folta4, Marja Rantanen1,
Lars Paulin5, Petri Auvinen5 and Paula Elomaal


Address: 'Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Helsinki, Finland, 2Finnish Graduate School in Plant
Biology, PO Box 56, FIN-00014 University of Helsinki, Helsinki, Finland, 3Viikki Graduate School in Biosciences, PO Box 56, FIN-00014
University of Helsinki, Helsinki, Finland, 4Horticultural Sciences Department, University of Florida, Gainesville, FL, USA and 5Institute of
Biotechnology, PO Box 56, FIN-00014 University of Helsinki, Helsinki, Finland
Email: Katriina Mouhu katriina.mouhu@helsinki.fi; Timo Hyt6nen* timo.hytonen@helsinki.fi; Kevin Folta kfolta@ifas.ufl.edu;
Marja Rantanen marja.rantanen@helsinki.fi; Lars Paulin lars.paulin@helsinki.fi; Petri Auvinen petri.auvinen@helsinki.fi;
Paula Elomaa paula.elomaa@helsinki.fi
* Corresponding author tEqual contributors



Published: 28 September 2009 Received: 10 December 2008
BMC Plant Biology 2009, 9:122 doi:10.1186/1471-2229-9-122 Accepted: 28 September 2009
This article is available from: http://www.biomedcentral.com/1471-2229/9/122
2009 Mouhu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.ore/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: We are studying the regulation of flowering in perennial plants by using diploid wild
strawberry (Fragaria vesca L.) as a model. Wild strawberry is a facultative short-day plant with an obligatory
short-day requirement at temperatures above 15C. At lower temperatures, however, flowering
induction occurs irrespective of photoperiod. In addition to short-day genotypes, everbearing forms of
wild strawberry are known. In 'Baron Solemacher' recessive alleles of an unknown repressor, SEASONAL
FLOWERING LOCUS (SFL), are responsible for continuous flowering habit. Although flower induction has a
central effect on the cropping potential, the molecular control of flowering in strawberries has not been
studied and the genetic flowering pathways are still poorly understood. The comparison of everbearing
and short-day genotypes of wild strawberry could facilitate our understanding of fundamental molecular
mechanisms regulating perennial growth cycle in plants.
Results: We have searched homologs for 118 Arabidopsis flowering time genes from Fragaria by EST
sequencing and bioinformatics analysis and identified 66 gene homologs that by sequence similarity,
putatively correspond to genes of all known genetic flowering pathways. The expression analysis of 25
selected genes representing various flowering pathways did not reveal large differences between the
everbearing and the short-day genotypes. However, putative floral identity and floral integrator genes API
and LFYwere co-regulated during early floral development. API mRNA was specifically accumulating in the
shoot apices of the everbearing genotype, indicating its usability as a marker for floral initiation. Moreover,
we showed that flowering induction in everbearing 'Baron Solemacher' and 'Hawaii-4' was inhibited by
short-day and low temperature, in contrast to short-day genotypes.
Conclusion: We have shown that many central genetic components of the flowering pathways in
Arabidopsis can be identified from strawberry. However, novel regulatory mechanisms exist, like SFL that
functions as a switch between short-day/low temperature and long-day/high temperature flowering
responses between the short-day genotype and the everbearing 'Baron Solemacher'. The identification of
putative flowering gene homologs and API as potential marker gene for floral initiation will strongly
facilitate the exploration of strawberry flowering pathways.




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Background
Transition from vegetative to reproductive growth is one
of the most important developmental switches in plant's
life cycle. In annual plants, like Arabidopsis, flowering and
consequent seed production is essential for the survival of
the population until the following season. To assure
timely flowering in various environments, Arabidopsis uti-
lizes several genetic pathways that are activated by various
external or internal cues. Light and temperature, acting
through photoperiod, light quality, vernalization and
ambient temperature pathways, are the most important
environmental factors regulating flowering time [1].
Moreover, gibberellin (GA) and autonomous pathways
promote flowering by responding to internal cues [2,3 ]. In
contrast to annual plants, the growth of perennials contin-
ues after generative reproduction, and the same develop-
mental program is repeated from year to year. Regulation
of generative development in these species is even more
complex, because other processes like juvenility, winter
dormancy and chilling are tightly linked to the control of
flowering time.

In Arabidopsis photoperiodic flowering pathway, phyto-
chrome (phy) and cryptochrome (cry) photoreceptors
perceive surrounding light signals and reset the circadian
clock feedback loop, including TOC1 (TIMING OF CAB
EXPRESSION), CCA1 (CIRCADIAN CLOCK ASSOCI-
ATED 1) and LHY (LATE ELONGATED HYPOCOTYL) [4-
7]. The central feature in the photoperiodic flowering is
the clock generated evening peak of CO (CONSTANS)
gene expression [8]. In long-day (LD) conditions, CO
peak coincidences with light resulting in accumulation of
CO protein in the leaf phloem and consequent activation
of the expression of FT (FLOWERING LOCUS T) [9]. FT
protein, in turn, moves to the shoot apex, and together
with FD triggers floral initiation by activating floral iden-
tity gene AP1 (APETALA 1) [10,11]. FT, together with
SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CON-
STANS 1) and LFY (LEAFY) form also convergence points
for different flowering pathways, and therefore are called
flowering integrator genes [12].

In winter-annual ecotypes of Arabidopsis, MADS-box gene
FLC (Fi .i-.. ii, Locus C) prevents flowering by repressing
FT and SOC1, and vernalization is needed to nullify its
function [13]. The major activator of FLC is FRI (FRIG-
IDA) [14], but several other proteins, including for exam-
ple FRL1 (FRIGIDA-LIKE 1) [15], PIE (PHOTOPERIOD
INDEPENDENT EARLY FLOWERING 1) [16], ELF7 and
ELF8 (EARLY FLOWERING 7 and 8) [17], and VIP3 (VER-
NALIZATION INDEPENDENCE 3) [18] are also needed
to maintain high FLC expression. During vernalization,
FLC is down-regulated by VRN2-PRC2 (Vernalization 2 -
Polycomb Repressive Complex 2) protein complex con-
taining low temperature activated VIN3 (VERNALIZA-
TION INSENSITIVE3), allowing plants to flower [19,20].


Autonomous and GA pathways respond to endogenous
cues to regulate flowering time. The role of the autono-
mous pathway is to promote flowering by lowering the
basal level of FLC transcription [3]. Autonomous pathway
consists of few sub-pathways, which include for example
RNA processing factors encoded by FCA, FPA, FLK
(FLOWERING LOCUS K), FY and LD (LUMINIDEPEND-
ENS) [21], putative histone demethylases LDL1 and LDL2
(LSD1-LIKE 1 and 2) [22], and deacetylases FLD (Flower-
ing locus D) and FVE [23,24]. GA pathway is needed to
induce LFY transcription and flowering in short-day (SD)
conditions [25].

Strawberries (Fragaria sp.) are perennial rosette plants,
belonging to the economically important Rosaceae fam-
ily. Most genotypes of garden strawberry (Fragaria x anan-
assa Duch.) and wild strawberry (F. vesca L.) are
Junebearing SD plants, which are induced to flowering in
decreasing photoperiod in autumn [26,27]. In some gen-
otypes, flowering induction is also promoted by decreas-
ing temperatures that may override the effect of the
photoperiod [27,28]. In contrast to promotion of flower-
ing by decreasing photoperiod and temperature, these
"autumn signals" have opposite effect on vegetative
growth. Petiole elongation decreases after a few days, and
later, around the floral transition, runner initiation ceases
and branch crowns are formed from the axillary buds of
the crown [29,30]. Crown branching has a strong effect on
cropping potential as it provides meristems that are able
to initiate inflorescences [31].

In addition to SD plants, everbearing (EB) genotypes are
found in garden strawberry and in wild strawberry
[29,32]. Environmental regulation of induction of flower-
ing in EB genotypes has been a topic of debate for a long
time. Several authors have reported that these genotypes
are day-neutral [29,33]. Recent findings, however, show
that long-day (LD) accelerates flowering in several EB Fra-
garia genotypes [34,35]. Interestingly, in wild strawberry
genotype 'Baron Solemacher' recessive alleles of SFL gene
locus (SEASONAL FLOWERING LOCUS) have been
shown to cause EB flowering habit [36]. SFL has not been
cloned, but it seems to encode a central repressor of flow-
ering in wild strawberry. Consistent with the repressor
theory, LD grown strawberries have been shown to pro-
duce a mobile floral inhibitor that is able to move from
mother plant to the attached runner plant [37]. GA is one
candidate corresponding to this inhibitor, since exoge-
nously applied GA has been shown to repress flowering in
strawberries [38,39].

Identification of central genes regulating flowering time
and EB flowering habit, as well as those controlling other
processes affecting flowering, is an important goal that
would greatly accelerate breeding of strawberry and other
soft fruit and fruit species of Rosaceae family. In this

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paper, we have searched Fragaria homologs with the
known Arabidopsis flowering time genes by EST sequenc-
ing and bioinformatics analysis. Dozens of putative flow-
ering genes corresponding to all known genetic pathways
regulating flowering time were identified. The expression
analysis of several candidate flowering time genes
revealed only few differences between the SD and EB wild
strawberries, including the presence or absence of AP1
mRNA in the apices of EB and SD genotypes, respectively.
Our data provides groundwork for detailed studies of
flowering time control in Fragaria using transcriptomics,
functional genomics and QTL mapping.

Results
Environmental regulation of flowering in two EB
genotypes of wild strawberry
We studied the effect of photoperiod and temperature on
flowering time in two EB genotypes, 'Baron Solemacher',
which contains recessive alleles in SFL locus [40,41], and
'Hawaii-4'. Flowering time was determined by counting
the number of leaves in the main crown before formation
of the terminal inflorescence. In SD genotypes of the wild
strawberry, SD (<15 h) or, alternatively, low temperature
(~10C) is needed to induce flowering [271. In EB geno-
types 'Baron Solemacher' and 'Rugen', instead, LD and
high temperature has been shown to accelerate generative
development [351, but careful analysis of the environ-
mental regulation of flowering induction has so far been
lacking.

Both 'Baron Solemacher' and 'Hawaii-4' produced five to
six leaves in LD at 18 oC before the emergence of the ter-
minal inflorescence showing that they are very early-flow-
ering in favorable conditions (Figure 1A and 1B). In
'Baron Solemacher', low temperature (11 C) or SD treat-
ment for five weeks at 18 o C clearly delayed flowering, but
low temperature did not have an additional effect on
flowering time in SD. Also in 'Hawaii-4', SD and low tem-
perature delayed flowering, but all treatments differed
from each other. Compared to the corresponding LD
treatment, SD at 18 o C doubled the number of leaves, and
low temperature (11 C) delayed flowering time by about
three leaves in both photoperiods. Thus, flowering induc-
tion in these EB genotypes is oppositely regulated by pho-
toperiod and temperature than previously shown for the
SD genotypes [27].

Construction and sequencing of subtracted cDNA libraries
We constructed two subtracted cDNA libraries from LD
grown EB genotype 'Baron Solemacher' and SD genotype,
in order to identify differentially expressed flowering time
genes in these genotypes. Plants were grown in LD condi-
tions, where the SD genotype stays vegetative and the EB
plants show early flowering. Pooled shoot apex sample
covering the floral initiation period was collected from the
EB genotype, and vegetative apices of the same age were


14 -
12- A
10 -
8 -
6-

4- SD11 SD18
SD11 OC SD180C


LD11C


20 B
B
S15
10-
S5-
0-


SD11C


SD18C


LD11C


LD18oC









LD18oC


Figure I
Environmental regulation of flowering in everbearing
wild strawberries. The effect of photoperiod (SD 12 h, LD
18 h) and temperature (I 1/18C) on the flowering time of
'Baron Solemacher' (A) and 'Hawaii-4' (B). Seeds were germi-
nated in LD at 18C, and seedlings were exposed to the
treatments for five weeks, when the cotyledons were
opened. After treatments, plants were moved to LD at 18C
and flowering time was recorded as number of leaves in the
main crown before the terminal inflorescence. Values are
mean SD. Pairwise comparisons between the treatments
were done by Tukey's test, and statistically significant differ-
ences (p _< 0.05) are denoted by different letters above the
error bars.


sampled from the SD genotype. Suppression subtractive
hybridization (SSH), the method developed for extraction
of differentially expressed genes between two samples
[42], was used to enrich either flowering promoting or
flowering inhibiting transcripts from EB and SD geno-
types, respectively.

A total of 1172 ESTs was sequenced from the library
enriched with the genes of the SD genotype (SD library
subtracted with EB cDNA) and 1344 ESTs from the library
enriched with the EB genes (EB library subtracted with
cDNA of the SD genotype). 970 SD ESTs [Gen-
bank:CH202443-GH2034121 and 1184 EB ESTs [Gen-
Bank:GH201259-GH202442] passed quality checking.
Pairwise comparison of these EST datasets revealed that
there was very little overlap between the libraries. How-
ever, general distribution of the sequences to functional
categories (FunCat classification) did not reveal any major
differences between the two libraries (Additional file 1).

BLASTx searches against Arabidopsis, Swissprot and non-
redundant databases showed that over 70% of the ESTs
gave a match in one or all of the three databases (Table 1).
Moreover, tBLASTx comparison with different genomes

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Table I: The comparison of F. vesca ESTs with different databases.


number


A) Raw
Poor Quality
Singletons/ESTs




B) Arabidopsis
Swissprot
Non-redundant
In all 3 datab.

C) Malus
Oryza
Vitis
Populus

D) No protein hits
No Fragaria hits


average length


number


number

1344
160
1184

number

781
570
852
862

874
807
761
928

322
454


average length


Average numbers, lengths and percentages of ESTs from EB and SD genotypes. A) numbers and average lengths of raw and poor quality ESTs, and
singletons, B) numbers and percentages of BLASTx hits against protein databases, C) numbers and percentages of tBLASTx hits against TIGR plant
transcript assemblies of Malus x domestic, Oryza sativa and Vitis vinifera and against Populus genome database, D) numbers and percentages of novel
ESTs.


revealed highest number of hits with Populus trichocarpa
(Table 1). We also performed tBLASTx searches against
TIGR plant transcript assemblies of Malus x domestic,
Oryza sativa and Vitis vinifera and found hits for 64-76%
of ESTs in these assemblies. Finally, the comparison of our
sequences with a current Fragaria unigene list at the
Genome Database for Rosaceae (GDR) showed that
38.2% of our ESTs are novel Fragaria transcripts. Taken
together, depending on the analysis, 15-22% of sequences
from SD genotype and 22-27% of EB sequences encode
novel proteins, or originate from untranslated regions of
mRNA. Moreover, the high number of novel Fragaria
sequences in our libraries indicates that SSH method effi-
ciently enriched rare transcripts in the libraries.

Identification of flowering time genes
Flowering related genes were identified from our libraries
by BLASTx searches as described above and fourteen puta-
tive flowering time regulators were identified; four gene
homologs were present only in EB library, eight in SD
library, and two genes in both libraries. In figure 2, we
have summarized the Arabidopsis flowering pathways and
highlighted the putative homologous genes identified
from our EST collection. In general, candidate genes for all
major pathways were identified. In addition, 118 Arabi-
dopsis flowering time genes were used as a query to search
publicly available GDR Fragaria EST and EST contig data-
bases using tBLASTn. Sequences passing cut-off value of


gibberellin photoperiod light quality


autonomous vernalization


TOC1


ELF6| |YCCA1PP3

\ |PRR7





GA C
signalinq FAP2 co HiRs1 P-Fi 11 Fs vPi


FSUF4|
FRL1
FRI

VIP3


Figure 2
A simplified chart showing Arabidopsis flowering
pathways and corresponding gene homologs in Fra-
garia. Gene homologs found in cDNA libraries produced
from SD and EB genotypes are surrounded by blue and red
boxes, respectively. Arrows indicate positive regulation and
bars negative regulation.




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le-10 were further analysed by BLASTx algorithm against
Arabidopsis protein database, and those returning original
Arabidopsis protein were listed. Moreover, sequences that
were absent from Fragaria databases were similarly
searched from GDR Rosaceae EST database. In these
searches, 52 additional Fragaria sequences were identi-
fied. Moreover, the total number of 88 homologs of Ara-
bidopsis flowering time genes were found among all
available Rosaceae sequences (Additional file 2).

Most genes of the Arabidopsis photoperiodic pathway were
found also in Fragaria, and some of the lacking genes were
present among Rosaceae ESTs (Table 2, Additional file 2).


We found several genes encoding putative Fragaria pho-
toreceptor apoproteins including phyA, phyC, cry2, ZTL
(ZEITLUPE) and FKF1 FLAVINN BINDING KELCH
REPEAT F-BOX 1) [43]. Of the central circadian clock
genes, homologs of LHY and TOC1 [5,7] were present in
our EST libraries and GDR, respectively, but CCA1 [6] was
lacking from both Fragaria and Rosaceae databases. Fur-
thermore, a putative Fragaria CO from the flowering regu-
lating output pathway has been cloned earlier [44].
Among the regulators of CO transcription and protein sta-
bility, GI (GIGANTEA) [45] was identified from Rosaceae
and putative COP1, SPA3 and SPA4 [46,47] from Fragaria.
In addition to genes of the photoperiodic pathway,


Table 2: The list of genes belonging to the photoperiodic flowering pathway.


Gene AT gene locus

Photoreceptors and clock input


Biological function


Act./Repr. +/-


Reference Fragaria


Red light photoreceptor
Red light photoreceptor
Blue light photoreceptor
Blue light photoreceptor
F-box protein/blue light photoreceptor
F-box protein/blue light photoreceptor
Unknown
Ser/Thr-specific protein phosphatase 2A
Unknown




Myb domain TF
Myb domain TF
Pseudo-response regulator
Myb TF
Unknown
Unknown
Pseudo-response regulator
Pseudo-response regulator
Jumonji/zinc finger-class TF




putative zinc finger TF

Phosphatidylethanolamine binding
Phosphatidylethanolamine binding
bZIP TF
E3 ubiquitin ligase
WD domain protein
WD domain protein
WD domain protein
Ring domain zinc finger
CCAAT-binding TF


VES-002-C06
nf
nf
DY669844
EX668764
DY671170
DY675323
BAR-009-A02
C0817759


VES-005-E09
nf
DY673134
DY668516
EX674323
nf
DY676242
VES-013-D 12
VES-002-F05


+ [8]
[65]
+ [I1]
[87]
+ [10]
[46]
[47]
[47]
[47]
[88]
+ [89]


DY672035
nf
nf
nf
EX675574
DY667888
nf
DY671873
DY671245
nf
EX658204


5E-33


2E-1 10
2E-97
2E-54
3E-33
I E-56
IE-10


9E-19

I E-75
3E-43
2E-25

3E-56
5E-52
I E-45


2E-45



2E-14
I E-94

3E-24
2E-83

2E-60


The most important genes belonging to the photoperiodic pathway in Arabidopsis and their biological function are presented. Floral activators and
repressors are indicated by + and marks, respectively. Moreover, the presence or absence of homologous sequence in Fragaria sequence
databases and E-value of BLASTx comparison against Arabidopsis are indicated. Sequences found in our libraries are named BAR and VES for
everbearing genotype 'Baron Solemacher' and short-day genotype, respectively. Other ESTs and EST contigs are found from Genome Database for
Rosaceae http://www.bioinfo.wsu.edu/gdr/. More complete list is available in Additional file 2.


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PhyA
PhyB
CRY I
CRY2
ZTL
FKFI
ELF3
FYPP3
SRRI


AT I G09570
AT2G 18790
AT4G08920
AT I G04400
AT5G57360
AT I G68050
AT2G25920
AT I G50370
AT5G59560


E-value


Circadian clock


LHY
CCA I
TOCI
LUX
ELF4
GI
PRR5
PRR7
ELF6


AT I GO 1060
AT2G46830
AT5G61380
AT3G46640
AT2G40080
AT I G22770
AT5G24470
AT5G02810
AT5G04240


Output pathway


CO
CDFI
FT
TFLI
FD
COPI
SPA I
SPA3
SPA4
RFI2
HAP3b


AT5G 15840
AT5G62430
AT I G65480
AT5G03840
AT4G35900
AT2G32950
AT2G46340
AT3G 15354
ATIG53090
AT2G47700
AT5G47640


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homologs for both known sequences belonging to light
quality pathways, PFT1 (PHYTOCHROME AND FLOW-
ERING TIME 1) and HRB1 (HYPERSENSITIVE TO RED
AND BLUE 1) [48,49], were found from our EST libraries.

For the vernalization pathway, we were not able to find
FLC-like sequences from our EST libraries or public Fra-
garia or Rosaceae EST databases by tBLASTn searches
although we used the FLC and FLC-like sequences from
Arabidopsis (MAF1-MAF5, MADS AFFECTING FLOWER-
ING 1-5) and several other plant species as query
sequences [13,50,51]. Similarly, also FRI [14] was lacking
from Rosaceae ESTs but putative FRL (FRIGIDA-LIKE)
[15] sequences were identified in Fragaria. In addition, we


identified several gene homologs belonging to the FRI
complex as well as other regulatory complexes (SWR1,
PAF) involved in promoting the expression of FLC (Table
3, Additional file 2) [17,52,53]. Also putative members of
FLC repressing PRC2 complex, were present in strawberry
ESTs. These include putative VIN3 (VERNALIZATION
INSENSITIVE 3) [19,20] that has been identified earlier
[541, and putative SWN1 (SWINGER 1), FIE (FERTILIZA-
TION INDEPENDENT ENDOSPERM), VRN1 (VERNALI-
ZATION 1) and LHP1 (LIKE HETEROCHROMATIN
PROTEIN 1) [19,55,56], which were found in this investi-
gation (Table 3, Additional file 2). However, putative
VRN2 that is needed for the repression of FLC by PRC2
was not found [19].


Table 3: The list of genes belonging to the vernalization pathway.


AT gene locus


Biological function


Act./Repr. +/-


Reference Fragaria


FLC AT5G10140 MADS-boxTF
MAFI/FLM ATIG77080 MADS-box TF

Fri complex

FRI AT4G00650 Unknown, enhancer of FLC
FRL I AT5G 16320 Unknown, enhancer of FLC
FRL2 AT I G31814 Unknown, enhancer of FLC
FESI AT2G33835 CCCH zinc finger protein
SUF4 ATI G30970 putative zinc finger containing TF

Swr complex

PIE AT3G 12810 ATP-dependent chromatin-remodelling factor
SEFI/SWC6 AT5G37055 Component of chromatin remodelling complex
ARP6/ESD I AT3G33520 Component of chromatin remodelling complex
ATXI AT2G31650 Putative SET domain protein


Pafl complex

ELF7
ELF8
VIP4
VIP3
EFS/SDG8

VRN2-PRC2 co.

VRN2
CLF
SWN I/EZA
FIE
VIN3
LHPI
VRNI


ATI G79730
AT2G06210
AT5G61150
AT4G29830
ATI G77300

mplex

AT4G 16845
AT2G23380
AT4G02020
AT3G20740
AT5G57380
AT5G 17690
AT3G 18990


nf
EX686406
Contig 4768
nf
BAR-003-F06


nf
DY670674
nf
EX687477


RNA polymerase 2 associated factor I -like
RNA polymerase 2 associated factor -like
RNA polymerase 2 associated factor -like
RNA polymerase 2 associated factor -like
putative histone H3 methyltransferase


nf
BAR-008-H08
EX660943
EX675781 I
nf




nf
nf
EX687655
DY671601
C0816801
DY669633
DY670727


Polycomb group zinc finger
Polycomb group protein
Polycomb group protein
Polycomb group protein
PHD domain protein
epigenetic silencing
DNA binding protein


4E-45
6E-49

5E-46


4E-70

4E-71


3E-42
2E-50
7E-98







3E-1 14
IE-l 12
2E-58
2E-40
8E-43


The most important genes belonging to the vernalization pathway in Arabidopsis and their biological function are presented. Floral activators and
repressors are indicated by + and marks, respectively. Moreover, the presence or absence of homologous sequence in Fragaria sequence
databases and E-value of BLASTx comparison against Arabidopsis are indicated. Sequences found in our libraries are named BAR and VES for
everbearing genotype 'Baron Solemacher' and short-day genotype, respectively. Other ESTs and EST contigs are found from Genome Database for
Rosaceae http://www.bioinfo.wsu.edu/gdr/. More complete list is available in Additional file 2.


Page 6 of 16
(page number not for citation purposes)


Gene


E-value


BMC Plant Biology 2009, 9:122








http://www. biomedcentral.com/1471-2229/9/122


In addition to the photoperiod and the vernalization
pathways, we searched candidate genes for the autono-
mous and GA pathways. Several sequences corresponding
to Arabidopsis genes from both pathways were identified
suggesting the presence of these pathways also in Fragaria
(Table 4, Additional file 2). Among these genes we found
homologs for Arabidopsis FVE and SVP which have been
shown to control flowering in a specific thermosensory
pathway [24,57]. Moreover, some additional flowering
time regulators that are not placed to any specific pathway
were identified (Table 4, Additional file 2).

Identification of floral integrator genes in Fragaria
Sequencing of our EST collections did not reveal any
homologs for the floral integrator or identity genes such
as FT, SOC1, LFY or AP1 [12,58]. A full-length cDNA
sequence of SOC1 homolog [GenBank:F1531999] and a
713 bp 3'-end fragment of putative LFY [Gen-
Bank:F1532000] were isolated using PCR. Closest protein
homolog of the putative FvSOC1 was 72% identical Popu-


lus trichocarpa MADS5, and the putative FvLFY showed
highest amino acid identity (79%) to Malus domestic FL2.
Comparison to Arabidopsis showed that AtSOC1 and
AtLFY, respectively, were 66% and 75% identical with the
corresponding wild strawberry protein sequences (Figure
3A and 3B). FT homolog, instead, was not identified in
Fragaria despite of many attempts using degenerate PCR
and screening of cDNA library plaques and E.coli clones
from a variety of tissues and developmental conditions
with the Arabidopsis coding sequence (K. Folta, unpub-
lished). However, a putative FT was found in Prunus and
Malus protein databases at NCBI. Among the other genes
belonging to the same gene family, homologs of MFT
(MOTHER OF FT AND TFL1) and ATC (ARABIDOPSIS
CENTRORADIALIS) [59] were present in GDR Fragaria
EST. Moreover, an EST contig corresponding to the floral
identity gene AP1 was found. The length of the translated
protein sequence of FvAP1 was 284 amino acids, being 30
amino acids longer than the corresponding Arabidopsis
sequence. However, FvAP1 EST contig contained an


Table 4: The list of genes belonging to autonomous and gibberellin flowering pathways.


AT gene locus


Biological function


Act./Repr. +/-


Reference Fragaria


Autonomous pathway


FCA
FPA
FLK
FY
SKBI
FVE
LD
FLD
LDLI/SWPI
LDL2


AT4G 16280
AT2G43410
AT3G04610
AT5G 13480
AT4G31120
AT2G 19520
AT4G02560
AT3G 10390
ATI G62830
AT3G 13682


Gibberellin pathway


GAI
RGA
SPY
DDFI
DDF2
AtMYB33
FPFI


SVP
AP2
PFT I
HRBI


AT I G 14920
AT2G01570
AT3G 11540
AT IG 126 10
ATIG63030
AT5GO6100
AT5G24860


AT2G22540
AT4G36920
ATIG25540
AT5G49230


RRM-type RNA binding domain containing
RRM-type RNA binding domain containing
KH-type RNA binding domain containing
mRNA 3' end processing factor
Arginine methyltransferase
retinoblastoma associated
DNA/RNA binding homeodomain protein
component of histone deacetylase complex
Histone H3-Lys 4 demetylase-like
Histone H3-Lys 4 demetylase-like




putative transcriptional repressor
putative transcriptional repressor
O-linked N-acetylglucosamine transferase
AP2-like TF
AP2-like TF
MYB TF
Unknown




MADS-box TF
AP2 TF
vWF-A domain protein
ZZ type zinc finger protein


The most important genes of Arabidopsis autonomous and gibberellin pathways as well as some other floral regulators are presented. The biological
function of the genes is indicated, and floral activators and repressors are marked by + and marks, respectively. Moreover, the presence or
absence of homologous sequence in Fragaria sequence databases and E-value of BLASTx comparison against Arabidopsis are indicated. Sequences
found in our libraries are named BAR and VES for everbearing genotype 'Baron Solemacher' and short-day genotype, respectively. Other ESTs and
EST contigs are found from Genome Database for Rosaceae http://www.bioinfo.wsu.edu/gdr/. More complete list is available in Additional file 2.


Page 7 of 16
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Gene


E-value


nf
nf
EX668302
EX659635
nf
VES-001 -B03
DY670534
nf
Contig 2573
DY669828


5E-52
5E-75

3E-76
3E-49

2E-27
I E-42


3E-147
8E-60
2E-93
5E-49

5E-29
7E-38


5E-22
9E-16
I E- 17
7E-22


[100]
[100]
[101]
[102]
[102]
[25]
[103]




[57]
[104]
[48]
[49]


Contig 3276
DQ 195503
BAR-002-C02
Contig 3158
nf
DY669997
Contig 4074




VES-013-D05
VES-008-A07
BAR-002-D08
VES-012-B01


BMC Plant Biology 2009, 9:122







http://www. biomedcentral.com/1471-2229/9/122


MVRGTQVRIEATSRVTFKRRGLLKAFESILDAEALIISPRKLYFAS


F H ~


SMQETIER KHTRDNQMNM
SMQETIER RHIKENNTN
MQDTIDR RHTKD S
CTIEEL EQQLERS Til
CTIEELQQIEQQLERS Til
CSIEELQQIEQQLEKS TT,
R, I]]


QEQIEILKKELLAEIRLUDiCA]


L
H


ESSTSSDVEJELFIGLEIRSKH
ISS ICIa iK


GRGVQKRENINRVTSKRSLLKAHISLCAEALIFSKGLFYS
GRGRQLKIENKNRQTFSKRSGLKKAEISLCDAVALVFSTGKLEYH


-IEIEYRSYEQLNE


C DQC


SNWTLEVQ
TS i Ei A KaLKAiRa EVaLQ ahRNi-SHF


DQLM LNEQDAKVSKQLME ISIQKKLENLTKK
P N SKLNEQLSLKISKQVYSSEQKKAQQN QIKK


IINMPPP


3 H' E ET -
L' 0Y PMA-


Figure 3
Protein alignments of Fragaria flowering integrator and identity genes. Multiple alignments of Fragaria protein
sequences of full length SOCI (A), partial LFY (B) and full-length API (C) with closest protein homologs and corresponding
protein sequence of Arabidopsis thaliana. Alignments were done by ClustalW (A, B) or T-Coffee (C) and modified by Boxshade
program. F. vesca API protein sequence was translated from GDR Fragaria EST contig 4941. PTM5 = Populus tremuloides
MADS5, AFL2 = Apple FLORICAULA 2, PpAPI = putative Prunus persica API.



Page 8 of 16
(page number not for citation purposes)


E QMKIHESRLG
N QIKIHLVKRLG


fl=
Qm
cm


BMC Plant Biology 2009, 9:122


NGGGGGMLGERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQNIAKERGEK
ERGKINGLDYLFHLYNCIWLIQV IAK]IRGEK
GL ERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCREFLLQVIAKDRGEK
CPTKVTNQVFRYAK GASYINKPKMRHYVHCYALHCLDEEASNALRRAFKERGENVGAW
CPTKMTNIVFRYA]GAMINKPKMRHYVHCYALHCLDEEISNALRRI"RGDNIGAW
CPTKVTNQVFRYAKKSGASYINKPKMRHYVHCYALHCLDEEASNALRRAFKERGENVGIWI
QACYKPL IA-AGQ WDIDAIFNSHPRLSIWYVPTKLRQLCHAERNNATASSSASGG
QACY 41 IZZMGWDIDAIFQHPILSVWYVPTKLRQLCHAERNNATAS G
QACYKPTL.l TMGWDIDAVFNIHPRLSIWYVPTKLRQLCHJERNN I


I[KEKJAVA
EKAL
RE L


FQ77FQ-Mlo
WEQQVQNQG
!WDQQMNQ








http://www. biomedcentral.com/1471-2229/9/122


unknown sequence stretch of 81 bp at nucleotide position
596-677. Putative FvAP1 showed highest overall identity
(68%) with putative API from Prunus persica (Figure 3C).
Moreover, the 5' sequence containing 187 amino acids
(the sequence before the unknown part) was 73% identi-
cal with the Arabidopsis API1.

Gene expression analysis revealed few differences between
EB and SD genotypes
We compared the expression of selected flowering time
genes (Table 5) corresponding to each flowering pathway
in the leaf and shoot apex samples of EB and SD geno-
types in order to explore the role of different pathways.
Only few of the analysed genes were differentially
expressed between the genotypes. Floral integrator gene
LFYwas slightly up-regulated in the shoot apex samples of
EB (Table 6). Moreover, PCR expression analysis with two
different primer pairs showed that AP1 was specifically
expressed in EB apices correlating with the identity of the
meristems. Among the genes from different flowering
pathways, only two genes, vernalization pathway gene
ELF8 [17] and photoperiod pathway gene ELF3 [60], were
slightly differentially expressed between the genotypes
(Table 6).



Table 5: The list of PCR primers used in real-time RT-PCR.


Developmental regulation of floral integrator, floral
identity, and GA pathway genes
We analysed the developmental regulation of AP1, LFY,
SOC1, GA3ox and GA2ox transcription in the shoot apices
of LD grown plants of EB and SD genotype containing one
to four leaves. Ubiquitin, used as a control gene, was stable
between different developmental stages, but was ampli-
fied ~1 PCR cycle earlier in SD genotype (Additional file
3). Thus direct comparison between the genotypes is not
possible, but the trends during development are compara-
ble. Three genes, AP1, LFY, and GA3ox, had clear develop-
mental stage dependent expression pattern in EB apices,
showing biggest changes after one or two leaf stage (Figure
4). The expression of AP1 was detected in EB apices
already at one leaf stage, and its mRNA accumulated grad-
ually reaching 6-fold increase at two leaf stage and 50-fold
increase at four leaf stage (Figure 4A). In parallel, tran-
scription of LFY started to increase at 2-leaf stage, but the
change in its expression was much smaller (Figure 4B). A
floral integrator gene, SOC1, in contrast, did not show
clear developmental regulation (Figure 4C). Also GA
pathway was co-regulated with AP1 and LFY, since GA
biosynthetic gene GA3ox was strongly down-regulated
after two leaf stage (Figure 4D). In addition, GA catabo-
lism gene, GA2ox, tended to follow changes in the expres-


Gene Forward primer


Reverse primer


CAGACCAGCAGAGGCTTATCTT
CGGCATTACGTTCACTGCTA
CAGGTGAGGCGGATAGAGAA
CGCTCCAGAAGAAGGATAAGG
TCTGAAGCACGTAAGGTCTA
AAAGCTGGAGAAGGAGGCAGTC
TGCATGGGGTAGTGAAACAA
ACCCACATCGTTTGTGGTCT
TCCTCCAAGGAACAAGATGG
TTCGAAGGTCTTGGCAATGG
GACCGAGAAATCCACTCTGC
GACATCCACTCCGCCAAC
GCGACATGCCAAGGTTAGAATT
GAATGGTGGACATCAGCAATCC
ACAAAATGGCCCCTCATGTG
CGCTAGTCAAGGTCGAGGAG
GCTCAGAATGCTCCTCCTGT
AGCCCTTGATGTCATCAGCTG
TCTCCACACCTTTGATTGCCA
GGAGAGCCAGAACCAGGAG
GATCCAGCAGCAACCAAGTCTC
CGTGCTAAGGCAGATGAATGG
TGCGGTGTCAAATTGCATCA
CCTCACAATCATCCACCAATCC
CACCATGCCCAGAGCTTCA
TGCAGAAACAAACGAGTTCGG
CCCGAAATCCTTGATTGTTCC


TTCTGGATATTGTAGTCTGCTAGGG
CCTGTAACACGCCTGCATC
AGAGCTTTCCTCTGGGAGAGA
CATGTGACTGAGCCTGTGCT
ATCCTGATCATAACCTCCAG
CCGAGGATAAGGATTGCTTGGT
CACCTCCGACAGTGACCTTT
ACATCAGGATCCACCAGAGG
CCATTCCCCTGATTTGAGAG
GCGCCTGAGTTTTATCCAACAC
CTCTCCGTCCGACAAGTAGC
GTGGACCCCACCACTATCTG
TCAGCGCCTCACACTCTTACAC
CCTCCGAAAGAATTGCTCAACA
TGTGCTATGTGTCCATGGTGGT
CGACTTCATCTCCATCAGCA
TGAGTATTGCAGCCACTTGC
CCGATGAATGGTTGGCTAATG
ACACCATCAGTCTCCTGCCAAG
CTCACCTTCTTCCCCTTCCT
CCTCTTGGTGCAACAGAAGGAC
TGAAGCACACGGTCAAGACTTC
GGCAACACTCAAGATGGATTGC
CGCCGATGTTGATCACCAA
AGGCCAGAGGTGTTGTTGGAT
CCAAGAGCATCGATCATTTGGT
AACACTGCAATCGAACAACAGC


Tmvalue of the primers is 60 IC.


Page 9 of 16
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UBI
LFY
SOCI
API
API
LHY
ZTL
FKFI
ELF3
ELF6
COL4
CO
PFTI
HRB I
FYPP3
FRL
ELF8
VRN5
MSII
LHP I
FVE
SVP
SPY
GA3ox
GA2ox
TFL I
AP2


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Table 6: The expression of selected genes in the wild
strawberry.


MSII as a control FVE as a control


Shoot apex samples
API
LFY
ELF8
Leaf samples
ELF3


Expressed only in EB
1.8 0.4
1.5 0.1


1.5 0.1


Expressed only in EB
1.9 0.3
1.6 0.1


1.8 0.0


Relative gene expression in the shoot apex or leaf samples of LD
grown plants of EB genotype compared to SD genotype. Ct values of
genes of interest were normalized against Ctvalues of MSII and FVE
to get normalized ACt values. The expression ratios between
genotypes (EB/SD) were calculated from the formula 2ACtEB/2ActsD.
Values are mean standard deviation. Pooled shoot apex samples and
leaf samples at four leaf stage were used.

sion of GA3ox, although the results were not so clear (data
not shown). In SD genotype, in contrast, AP1 was absent
and other genes did not show clear developmental regula-
tion (Figure 4). In this experiment, control plants of EB


60 6
50 A B
40 4
30 3
20 2
0 0
1 2 3 4 1 2 3 4

2 25 D


o 0,5
S0 -
1 2 3 4 1 2 3 4


Number of leaves
---SD -A-EB


Number of leaves


Figure 4
Developmental regulation of gene expression in wild
strawberry shoot apices. The expression of API (A), LFY
(B), SOCI (C) and GA3ox (D) in the SD and EB ('Baron
Solemacher') genotype of the wild strawberry. Triplicate
shoot apex samples were collected from LD grown plants at
one to four leaf stage. Ct values were normalized against a
Ubiquitin [GenBank:DY672326] gene to get normalized ACt
values. The expression differences between one leaf stage
and later developmental stages were calculated from the for-
mula 2Act later developmental stage/2ACt one leaf stage. The expression
values at one leaf stage were artificially set to I separately for
both genotypes. Values are mean SD. Note that Ubiquitin
was amplified -I cycle earlier in SD genotype, but was stable
between different developmental stages. Therefore, expres-
sion values between genotypes cannot be directly compared,
while the expression levels between the various develop-
mental stages are comparable.


Gene


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genotype flowered very early, after producing 4.7 0.3
leaves to the main crown, whereas plants of SD genotype
remained vegetative.

Discussion
Identification of flowering genes in strawberry
Genetic regulation of flowering in strawberry has earlier
been studied only by crossing experiments. According to
Weebadde et al. [611, everbearing character is a polygenic
trait in garden strawberry whereas other studies indicate
the presence of a single dominant gene [62]. Different
results may arise from different origin of everbearing
habit, since at least three different sources have been used
in strawberry breeding [32,61,62]. Studies in F. vesca
'Baron Solemacher'have shown that EB flowering habit in
this genotype is controlled by recessive alleles of a single
locus, called seasonal flowering locus (sfl) [40,41]. Identifi-
cation of central genes regulating flowering, as well as
those controlling other processes that affect flowering
(runnering, chilling), is an important goal that would
greatly accelerate breeding of strawberry and other soft
fruit and fruit species of Rosaceae family.

For comprehensive identification of candidate genes of
the strawberry flowering pathways, we searched
homologs for 118 Arabidopsis flowering time genes from
our own cDNA libraries and from GDR. In total, we were
able to identify 66 gene homologs among about 53000
EST sequences. Moreover, gene homologs lacking from
Fragaria were further mined from Rosaceae EST collec-
tions containing about 410 000 EST sequences. These
searches revealed 22 additional putative flowering time
genes in Rosaceae. Ongoing genome sequencing projects
in apple, peach and wild strawberry will ultimately reveal
the currently lacking flowering regulators in these species
[631.

Sequences found in Fragaria corresponded to all known
Arabidopsis flowering time pathways [2] suggesting that all
of these genetic pathways may be present in Fragaria.
However, the sequence conservation does not necessarily
mean functional conservation, so major candidate genes
from different pathways have to be functionally character-
ized in order to prove the presence of these pathways in
strawberry. Few central regulators of flowering time are
lacking from Fragaria sequence collections and some of
them also from Rosaceae databases. For example, we were
not able to identify a homolog for the florigen gene FT
[11] in Fragaria regardless of several different attempts.
This is probably due to its low expression level and tissue
specific expression pattern [64]. Similarly, GI, which links
circadian clock and CO [8,65], was absent from the Fra-
garia sequences. FT and GI homologs were, however,
found in apple and Prunus, showing that they are present
in Rosaceae. Moreover, consistent with studies in model


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legumes [661, CCA1 was lacking in Rosaceae, but its
redundant paralog, LHY, is represented by few ESTs in Fra-
garia. CCA1 and LHY are MYB-type transcription factors
which repress the expression of TOC1 in the central loop
of Arabidopsis circadian clock [67]. Thus, in Fragaria and
other species of Rosaceae family, LHY alone may control
the expression of TOC1 in the clock core. This contrasts
with other species, like Populus, where duplications of the
LHY/CCA1 genes contribute to an apparently more com-
plex mode of clock control [68].

Vernalization pathway in Arabidopsis culminates in FLC
and FLC-like floral repressors [13,50]. They have been
functionally characterized only in Brassicaceae [13,69],
although homologous MADS box genes have been
recently found from several eudicot lineages by phyloge-
netic analysis [51]. However, we were not able to identify
FLC-like sequences in Rosaceae by using several FLC-like
sequences as a query. Similarly, also FRI homologs were
lacking from the Rosaceae sequence collections. However,
putative homologs of FRI-like genes, FRL1 and FRL2,
which are involved in FLC activation in Arabidopsis [15]
were found, as well as several other homologs of genes
belonging to FLC regulating protein complexes. Despite
the presence of these transcripts, the presence of FLC is
unclear, since at least PRC2 complex has several target
genes [70]. Cloning and characterization of putative FLC-
like and FRI genes as well as FT in strawberry would
greatly expand our understanding of strawberry flowering
pathways, and therefore, it is one of the most important
targets of further studies. If these transcripts are present in
strawberry, it is likely that the precise control of flowering
has placed their expression in specific tissues or contexts
where they are not easily detected. However, their pres-
ence should be substantiated in analysis of the impending
genome sequence. Another important goal is the identifi-
cation of putative Rosaceae or Fragaria specific flowering
time genes. Ultimately, transcriptomics studies and func-
tional analysis of central genes may reveal how different
flowering pathways, which may be closely related to Ara-
bidopsis pathways, make seasonal flowering in strawberry.

What is the SFL gene?
SFL is a single dominant locus that enforces seasonal
flowering habit in wild strawberry, and homozygous
mutation in this locus leads to continuous flowering habit
in at least one genotype, 'Baron Solemacher' [36]. In SD
genotypes of wild strawberry, SD or low temperature
induce flowering [27] probably by overcoming the func-
tion of SFL repressor gene. We showed here that EB geno-
types 'Baron Solemacher' and 'Hawaii-4' produce only 5 -
6 leaves to the main crown before the formation of the ter-
minal inflorescence in LD at 18C. Hence, flowering
induction in these conditions occurs soon after germina-
tion. In SD (12 h) or at low temperature (11 C) instead,


plants formed several leaves more before the inflores-
cence. This finding shows that, in contrast to SD geno-
types, both SD and low temperature restrain flowering
induction in these genotypes, confirming earlier sugges-
tions that EB genotypes of wild strawberry are in fact LD
plants [35]. Most simple explanation for these opposite
environmental responses is that the lack of flowering
inhibitor, produced by active SFL gene, unmasks LD
induced flowering promotion pathway in 'Baron
Solemacher' and possibly in other EB genotypes. Given
that both SD and low temperature repress SFL, analogous
flowering regulating pathway has not yet been character-
ized at molecular level.

Our gene expression analysis did not give any hints of the
putative location of SFL in wild strawberry flowering
pathways. However, homologs of certain flowering
repressors can be consireded as candidates for SFL, includ-
ing the rice CO homolog HD1 (HEADING DATE 1), or
Arabidopsis vernalization pathway genes FLC and FRI
[13,14,71]. In strawberry, the role of vernalization path-
way remains unclear until the presence or absence of FRI
or FLC function is confirmed or other targets for this path-
way are found. Strawberry CO, instead, has been cloned
and mapped in Fragaria reference map, but its position
does not match with the genomic location of SFL showing
that CO itself is not SFL [44,72]. However, the possibility
that some regulator of CO transcription or protein stabil-
ity could be SFL cannot be ruled out and should be stud-
ied further.

Exogenously applied GA inhibits flowering in wild straw-
berry, and therefore, GA has been suggested to be a floral
repressor [38,39]. Similar patterns have been observed
and delineate differences between recurrent and non-
recurrent roses [73]. However, we did not find clear differ-
ences in the expression of GA biosynthetic and catabolism
genes, GA3ox and GA2ox, in the shoot apex samples of EB
and SD genotypes before the floral initiation had
occurred. In contrast, GA3ox was strongly repressed in EB
apices after floral initiation and GA2ox showed similar
trend. The fact that these changes in GA pathway occurred
after two leaf stage suggests that GA signal was regulated
during early flower development rather than during floral
transition. These data does not support the role of endog-
enous GA as the regulator of flowering induction, indicat-
ing that SFL is not situated in the GA pathway. However,
quantitative analysis of GA levels is needed to show
whether the observed changes in the expression of GA
pathway genes are reflected at the metabolic level.

AP I is a potential marker of floral initiation in strawberry
Gene expression analysis revealed that two putative flow-
ering genes, AP1 and LFY, were co-regulated during floral
development in EB wild strawberry. The homolog of floral


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identity gene AP1 was expressed in the EB apex already at
one leaf stage, and its expression was strongly enhanced
during later developmental stages. Also LFY mRNA accu-
mulated along with AP1 during floral development in EB
genotype, whereas SOC1 did not show a clear trend. The
mRNA of SOC1 and LFY were present also in SD geno-
type, but AP1 transcription was not detected. In Arabidop-
sis, LFY and API activate each other's expression
constituting a feedback loop [12,58]. Moreover, AP1 is
activated by FT-FD heterodimer shortly after flowering
induction [10]. Thus, the expression patterns of APland
LFY in the meristems of EB genotype suggest that flower-
ing induction in these plants occurs before two leaf stage
in LD conditions. Consistent with this conclusion, flower
initials were clearly visible by stereomicroscope in the
meristems at three or four leaf stage, and plants flowered
after producing on average 4.7 leaves in the main crown.
Based on our results, AP1 can be used as a marker for flo-
ral initiation in wild strawberry. However, functional
studies are needed to confirm the role of AP1, LFY and
SOC1 as floral integrator and identity genes, and this
approach is currently going on.

Conclusion
We have explored putative components for the genetic
flowering pathways in perennial SD plant wild strawberry
by identifying 66 homologs of Arabidopsis flowering time
genes. Although few central genes are lacking, these data
indicate that all known genetic flowering pathways may
be present in Fragaria. This is consistent with the finding
that EB genotypes, 'Hawaii-4' and 'Baron Solemacher',
show similar environmental regulation of flowering than
Arabidopsis summer-annuals. We also studied the expres-
sion of selected candidate genes and found that few genes
were co-regulated in the shoot apex of the EB genotype
during early floral development. Most strikingly, the
mRNA of AP1 specifically accumulated in EB genotype,
but was absent in SD genotype, showing its usefulness as
a marker of floral initiation. Finally, identification of
putative flowering time genes reported here enables their
transcriptional and functional characterization, as well as
genetic mapping, which may give answers for the relative
importance of each genetic flowering pathway and lead to
cloning of the central repressor gene, SFL. Ultimately,
these genetic resources could be utilized in cultivar breed-
ing of various species of Rosaceae family through genetic
transformation and marker assisted selection breeding.

Methods
Plant materials, growing conditions and sampling
Seeds of SD and EB ('Baron Solemacher') genotypes of the
wild strawberry (NCGR accession numbers [PI551792]


and [PI551507], respectively) were sown on potting soil
mixture (Kekkila, Tuusula, Finland) and grown in a green-
house under LD conditions (day length min. 18 h), pro-
vided by 400 W SON-T lamps (Airam, Kerava, Finland)
and natural sunlight. After two to three leaves had devel-
oped per plant, shoot apex samples (tip of the shoot con-
taining the meristem as well as two to three leaf initials)
were collected under a stereomicroscope at ten different
time points with three days intervals. Samples from each
time point were pooled and used for the construction of
cDNA libraries and real-time RT-PCR. WT samples con-
tained shoot apices of the main crown, collected from 50
plants per time point. Also in EB genotype, shoot apices of
the main crown were collected until the sepal initials
became visible in the meristems. After this time point, the
apices from one to three side shoots per plant were col-
lected, altogether from 40 plants per sampling. In addi-
tion, leaf samples were collected from the same plants at
four leaf stage for real-time RT-PCR analysis. Moreover,
separate shoot apex samples were collected from WT and
EB genotypes at one, two, three and four leaf stages. Con-
trol plants were grown in LD and their flowering time was
determined by counting the number of leaves in the main
crown before the terminal inflorescence. All samples were
collected in July August 2006 2008.

Preparation and sequencing of subtracted cDNA libraries
Total RNA from pooled shoot apex samples was extracted
with a pine tree method for RNA isolation [74]. The cDNA
was synthesized with BD SMART cDNA Synthesis kit
(Clontech, Palo Alto, US), amplified with PCR as
instructed for subtraction, purified with Chroma Spin-
1000 DEPC-H20 Columns (Clontech), extracted with
chloroform:isoamylalcohol (24:1) using Phase Loch Gel
Heavy 1.5 ml tubes (Eppendorf, Hamburg, Germany),
digested with Rsal (Boehringer Mannheim, Mannheim,
Germany), and purified with High Pure PCR Product
Purification kit (Roche Diagnostics, Indianapolis, US).
The cDNAs were subtracted using BD PCR-Select cDNA
Subtraction Kit (Clontech) in both forward and reverse
directions. The forward and reverse PCR mixtures were
digested with Rsal (Boehringer Mannheim) and purified
with High Pure PCR Product Purification Kit (Roche).
After digestion, A-tailing was done as instructed in the
technical manual of pGEM-T and pGEM-T Easy Vector
Systems and PCR mixtures were ligated to pGEM-T Easy
Vector (Promega, Wisconsis, US), and electroporated to
TOP 10 cells. The libraries were sequenced at the Institute
of Biotechnology, University of Helsinki, as described ear-
lier [75].








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Bioinformatics analysis
Raw EST sequences were quality checked before annota-
tion. Base calling, end clipping and vector removal were
performed by CodonCodeAligner-software (CodonCode
Corporation, US). After this the ESTs were manually
checked and sequences that contained poly-T in the
beginning followed by short repetitive sequences were
removed. BLASTx was performed against functionally
annotated Arabidopsis protein database (v211200, MIPS),
Swissprot and non-redundant protein database (NCBI),
and Populus trichocarpa genome of DOE Joint Genome
Institute [76] using cut-off value le-10. tBLASTx was per-
formed against TIGR plant transcript assemblies of Malus
x domestic, Oryza sativa and Vitis vinifera [77], and GDR
Fragaria and Rosaceae Contigs using cut-off value le-10.
For MIPS BLAST hits corresponding functional classes and
Gene Ontology classes were obtained from Functional
Classification Catalogue (Version 2.1) and GO annota-
tion for Arabidopsis thaliana (Version 1.1213).

Homologs of Arabidopsis flowering time genes were
searched from GDR Fragaria contig and EST databases
using tBLASTx algorithm and Arabidopsis protein
sequences as a query. Homologous sequences passing a
cut-off value le-10 were further analysed by BLASTx algo-
rithm against Arabidopsis protein database, and sequences
showing highest sequence homology with the corre-
sponding Arabidopsis genes were selected. The sequences
lacking from Fragaria were similarly searched from GDR
Rosaceae EST database and from Rosaceae protein data-
base at NCBI.

Photoperiod and temperature treatments
For the analysis of environmental regulation of flowering
in EB genotypes, seeds of 'Baron Solemacher', and
'Hawaii-4' were germinated in 18 h LD at 18C. During
germination, plants were illuminated using 400 W SON-T
lamps (Airam) for 12 h daily (90 10 limol m-2 s-1 at plant
height plus natural light) and incandescent lamps were
used for low-intensity daylength extension (5 1 imol m-
2 s-1 at plant height). After opening of the cotyledons
plants were moved to four treatments, SD and LD (12/18
h) at low or high temperature (11/18 0C), for five weeks.
In LD, incandescent lamps were used for low-intensity
daylength extension (5 1 limol m-2 s-1 at plant height)
after 12 h main light period. Also photoperiods of 8 and
8 + 8 h (SD/LD) were tested, but because of very slow
growth in these light treatments, longer photoperiods
were selected (data not shown). SD treatments were car-
ried out at the greenhouse using darkening curtains, while
LD treatments (photoperiod 18 h) were conducted in a
similar greenhouse compartment without curtains. The
experiments were carried out during winter 2007 2008,
when the natural day length was under 12 h. After treat-
ments, plants were potted to 8 x 8 cm pots, moved to LD


(18 h), and flowering time was determined as described
above.

Gene expression analysis
Total RNA from leaf and shoot apex samples was extracted
with a pine tree method [74], and cDNAs were synthe-
sized from total RNA using Superscript III RT kit (Invitro-
gen, Carlsbad, US) and dT18VN anchor primers.
LightCycler 480 SYBR Green I Master kit (Roche Diagnos-
tics, Indianapolis, US) was used to perform 15 lil real-time
RT-PCR reactions in 384-well plates according to manu-
facturer's instructions by using Light Cycler 480 real-time
PCR system (Roche Diagnostics). PCR primers with Tm
value of 60 C were used (Table 5). Three biological repli-
cates were analysed for shoot apex samples from different
developmental stages (Figure 4), and two biological repli-
cates were used for pooled shoot apex and leaf samples
(Table 6).

Authors' contributions
TH, KM and PE designed all experiments. PE coordinated
the study and helped to draft the manuscript. TH run the
real-time PCR analysis, performed flowering gene
searches from sequence databases, and drafted the manu-
script together with KM. KM constructed the subtracted
cDNA libraries and performed bioinformatics analysis
together with KF. KF also helped to draft the manuscript.
MR participated in flowering time analysis and sampling
of shoot apices. PA and LP were responsible for the EST
sequencing. All authors read and approved the final man-
uscript.

Additional material


Additional file 1
Functional classification of ESTs from EB and SD genotypes. The per-
centage of gene hits ;,, Iii. .. FunCat classes in two cDNA libraries pre-
pared in this study is shown. Same gene may be classified in one or several
classes. WT and EB libraries were prepared from SD and everbearing gen-
otypes, respectively.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-9-122-S1.PDF]

Additional file 2
Complete list of flowering time genes searched. Genes belonging to dif-
i...,,, 11..... mi pathways are listed in separate sheets of .xls file. Homol-
ogous sequences found in Fragaria are indicated. Moreover, corresponding
Rosaceae sequences were searched, if Fragaria sequence was not found.
GenBank EST sequence number or Genome Database for Rosaceae contig
number is given for Fragaria and Rosaceae ESTs and contigs, respectively.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-9-122-S2.XLS]


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BMC Plant Biology 2009, 9:122


Additional file 3
The stability of control genes used in this study. Ct values of FVE, MSI
and UBI in the leaf samples collected at four leaf stage (a) and in the
pooled shoot apex samples (b). Same plant material of SD and EB ('Baron
Solemacher') genotypes was used than in Table 6. Panel c: Ct values of
UBI in the shoot apex samples of SD and EB genotypes 11 1h ... .1. I-
opmental stages. Values are means (+ standard deviation) of two (a, b)
or three (c) biological and three technical replicates. One g of total RNA
was used for cDNA synthesis for each sample. Di-i. .. Ct values of UBI
in shoot apex samples in figures b and c are due to ii, ..1 'NA dilu-
tions used for PCR.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-9-122-S3.PPT]




Acknowledgements
Dr. Michael Brosche is acknowledged for his kind help in the production of
subtracted cDNA libraries and M.Sc. Techn. Erkko Airo for his valuable
technical help. In addition, Finnish Ministry of Agriculture and Forestry is
thanked for financial support.

References
I. Ausin I, Alonso-Blanco C, Martinez-Zapater M: Environmental
regulation of flowering. IntJ Dev Biol 2005, 49:689-705.
2. Putterill J, Laurie R, Macknight R: It's time to flower: the genetic
control of flowering time. Bioessays 2004, 26:363-373.
3. Simpson GG: The autonomous pathway: epigenetic and post-
transcriptional gene regulation in the control of Arabidopsis
flowering time. Curr Opinion Plant Biol 2004, 7:570-574.
4. Imaizumi T, Kay SA: Photoperiodic control of flowering: not
only by coincidence. Trends Plant Sci 2006, I 1:550-558.
5. Strayer C, Oyama T, Schultz TF, Raman R, Somers DE, Mis P, Panda
S, Kreps JA, Kay SA: Cloning of the Arabidopsis clock gene
TOCI, an autoregulatory response regulator homolog. Sci-
ence 2000, 289:768-771.
6. Wang ZY, Tobin EM: Constitutive expression of the CIRCA-
DIAN CLOCK ASSOCIATED I (CCAI) gene disrupts circadian
rhytms and suppresses its own expression. Cell 1998,
93:1207-1217.
7. Schaffer R, Ramsay N, Samach A, Corden S, Putterill J, Carre IA, Cou-
pland G: The late elongated hypocotyl mutation of Arabidopsis
disrupts circadian rhythms and the photoperiodic control of
flowering. Cell 1999, 93:1219-1229.
8. Suirez-L6pez P, Wheatley K, Robson F, Onouchi H, Valverde F, Cou-
pland G: CONSTANS mediates between the circadian clock
and the control of flowering in Arabidopsis. Nature 2001,
410:1116-1120.
9. Yanovsky MJ, Kay SA: Molecular basis of seasonal time meas-
urement in Arabidopsis. Nature 2002, 419:308-312.
10. Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y,
Ichinoki H, Notaguchi M, Goto K, Araki T: FD, a bZIP protein
mediating signals from the floral pathway integrator FT at
the shoot apex. Science 2005, 309:1052-1056.
I I. Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis
A, Farrona S, Gissot L, Turnbull C, Coupland G: FT protein move-
ment contributes to long-distance signalling in floral induc-
tion of Arabidopsis. Science 2007, 316:1030-1033.
12. Parcy M: Flowering: a time for integration. Intj Dev Biol 2005,
49:585-593.
13. Searle I, He Y, Turck F, Vincent C, Fornara F, Krober S, Amasino RA,
Coupland G: The transcription factor FLC confers a flowering
response to vernalization be repressing meristem compe-
tence and systemic signalling in Arabidopsis. Genes Dev 2006,
20:898-912.
14. Johanson U, West Lister C, Michaels S, Amasino R, Dean C: Molec-
ular analysis of FRIGIDA, a major determinant of natural var-
iation in Arabidopsis flowering time. Science 2000,
290:344-347.


http://www. biomedcentral.com/1471-2229/9/122


15. Michaels SD, Bezerra IC, Amasino RM: FRIGIDA-related genes are
required for the winter-annual habit in Arabidopsis. PNAS
2004, 101:3281-3285.
16. Noh Y, Amasino RS: PIEI, an ISWI family gene, is required for
FLC activation and floral repression in Arabidopsis. Plant Cell
2003, 15:1671-1682.
17. He Y, Doyle MR, Amasino RM: PAF I-complex-mediated histone
methylation of FLOWERING LOCUS C chromatin is required
for the vernalization-responsive, winter-annual habit in Ara-
bidopsis. Genes Dev 2004, 18:2774-2784.
18. Zhang H, Ransom C, Ludwig P, van Nocker S: Genetic analysis of
early flowering mutants in Arabidopsis defines a class of plei-
otropic developmental regulator required for expression of
the flowering-time switch Flowering Locus C. Genetics 2003,
164:347-358.
19. Wood CC, Robertson M, Tanner G, Peacock WJ, Dennis ES, Helli-
well CA: The Arabidopsis thaliana vernalization response
requires a Polycomb-like protein complex that also includes
VERNALIZATION INSENSITIVE 3. PNAS 2006,
103:14631-14636.
20. Sung S, Amasino RM: Vernalization in Arabidopsis thaliana is
mediated by the PHD finger protein VIN3. Nature 2004,
427:159-164.
21. Quesada V, Dean C, Simpson GG: Regulated RNA processing in
the control of Arabidopsis flowering. Int J Dev Biol 2005,
49:773-780.
22. Jiang D, Yang W, He Y, Amasino RM: Arabidopsis relatives of the
human lysine-specific demethylase I repress the expression
of FWA and FLOWERING LOCUS C and thus promote the flo-
ral transition. Plant Cell 2007, 19:2975-2987.
23. He Y, Michaels SD, Amasino RM: Regulation of flowering time by
histone acetylation in Arabidopsis. Science 2003,
302:175 1-1754.
24. Ausin I, Alonso-Blanco C, Jarillo JA, Ruiz-Garcia L, Martinez-Zapater
JM: Regulation of flowering time by FVE, a retinoblastoma-
associated protein. Nat Genet 2004, 36:162-166.
25. Gogal GFW, Sheldon CC, Gubler F, Moritz T, Bagnall DJ, MacMillan
CP, Li SF, Parish RW, Dennis ES, Weigel D, King RW: GAMYB-like
genes, flowering, and gibberellin signaling in Arabidopsis. Plant
Physiol 2001, 127:1682-1693.
26. Jonkers H: On the flower formation, the dormancy and the
early forcing of strawberries. In Thesis Mededelingen van de
Landbouwhogeschool, Wageningen; 1965.
27. Heide 0, Sonsteby A: Interactions of temperature and pho-
toperiod in the control of flowering of latitudinal and altitu-
dinal populations of wild strawberry (Fragaria vesca). Physiol
Plant 2007, 130:280-289.
28. Heide 0: Photoperiod and temperature interactions in
growth and flowering of strawberry. Physiol Plant 1977,
40:21-26.
29. Guttridge CG: Fragaria x ananassa. In CRC Handbook of Flowering
Volume III. Edited by: Halevy A. Boca Raton: CRC Press; 1985:16-33.
30. Konsin M, Voipio I, Palonen P: Influence of photoperiod and
duration of short-day treatment on vegetative growth and
flowering of strawberry (Fragaria x ananassa Duch.). J Hort Sci
Biotech 2001, 76:77-82.
31. Hytonen T, Palonen P, Mouhu K, Junttila 0: Crown branching and
cropping potential in strawberry (Fragaria x ananassa, Duch.)
can be enhanced by daylength treatments. J Hort Sci Biotech
2004, 79:466-471.
32. Darrow GM: The strawberry. History, breeding and physiology New York:
Holt, Rinehart and Winston; 1966.
33. Durner EF, Barden JA, Himelrick DG, Poling EB: Photoperiod and
temperature effects on flower and runner development in
day-neutral, junebearing and everbearing strawberries. j
Amer Soc Hort Sci 1984, 109:396-400.
34. Sonsteby A, Heide OM: Long-day control of flowering in ever-
bearing strawberries. j Hort Sci Biotech 2007, 82:875-884.
35. Sonsteby A, Heide OM: Long-day rather than autonomous con-
trol of flowering in the diploid everbearing strawberry Fra-
garia vesca ssp. semperflorens. j Hort Sci Biotech 2008, 83:360-366.
36. Albani M, Battey NH, Wilkinson MJ: The development of ISSR-
derived SCAR markers around the SEASONAL FLOWERING
LOCUS (SFL) in Fragaria. Theor AppI Gen 2004, 109:571-579.





Page 14 of 16
(page number not for citation purposes)








http://www. biomedcentral.com/1471-2229/9/122


37. Guttridge CG: Further evidence for a growth-promoting and
flower-inhibiting hormone in strawberry. Annals Bot 1959,
23:612-621.
38. Thompson PA, Guttridge CG: Effect of gibberellic acid on the
initiation of flowers and runners in the strawberry. Nature
1959, 184:72-73.
39. Guttridge CG, Thompson PA: The effects of gibberellins on
growth and flowering of Fragaria and Duchesnea. J Exp Bot
1963, 15:631-646.
40. Brown T, Wareign PF: The genetic control of the everbearing
habit and three other characters in varieties of Fragaria
vesca. Euphytica 1965, 14:97-112.
41. Battey N, Miere P, Tehranifar A, Cekic C, Taylor S, Shrives K, Hadley
P, Greenland A, Darby J, Wilkinson M: Genetic and environmen-
tal control of flowering in strawberry. In Genetic and Environmen-
tal Manipulation of Horticultural Crops Edited by: Cockshull KE, Gray D,
Seymour GB, Thomas B. Wallingford, UK, Cab International;
1998:111-131.
42. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang
B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD:
Suppression subtractive hybridization: a method for gener-
ating differentially regulated or tissue-specific cDNA probes
and libraries. PNAS 1996, 93:6025-6030.
43. Thomas B: Light signals and flowering. J Exp Bot 2006,
57:3387-3393.
44. Stewart P: Molecular characterization of photoperiodic flow-
ering in strawberry (Fragaria sp.). In PhD thesis University of
Florida; 2007.
45. Fowler S, Lee K, Onouchi H, Samach A, Richardson K, Morris B, Cou-
pland G, Putterill J: GIGANTEA: a circadian clock-controlled
gene that regulates photoperiodic flowering in Arabidopsis
and encodes a protein with several possible membrane-
spanning domains. EMBOJ 1999, 18:4679-4688.
46. Jang S, Marchal V, Panigrahi KCS, Wenkel S, Soppe W, Deng X, Val-
verde F, Coupland G: Arabidopsis COP I shapes the temporal
pattern of CO accumulation conferring a photoperiodic
flowering response. EMBOJ 2008, 27:1277-1288.
47. Laubinger S, Marchal V, Le Gourrierec J, Wenkel S, Adrian J, Jang S,
Kulajta C, Braun H, Coupland G, Hoecker U: Arabidopsis SPA pro-
teins regulate photoperiodic flowering and interact with flo-
ral inducer CONSTANS to regulate its stability. Development
2006, 133:3213-3222.
48. Cerdcn PD, Chory : Regulation of flowering time by light qual-
ity. Nature 2003, 423:881-885.
49. Kang X, Zhou Y, Sun X, Ni M: HYPERSENSITIVE TO RED AND
BLUE I and its C-terminal regulatory function control
FLOWERING LOCUS T expression. Plant] 2007, 52:937-948.
50. Scortecci KC, Michaels SD, Amasino RM: Identification of a
MADS-box gene, FLOWERING LOCUS M, that repress flow-
ering. Plant] 2001, 26:229-236.
51. Reeves PA, He Y, Schmitz RJ, Amasino RM, Panella LW, Richards CM:
Evolutionary conservation of the FLOWERING LOCUS C-
mediated vernalization response: evidence from the sugar
beet (Beta vulgaris). Genetics 2007, 176:295-307.
52. Choi K, Park C, Lee J, Oh M, Noh B, Lee I: Arabidopsis homologs
of components of the SWRI complex regulate flowering and
plant development. Development 2007, 134:1931-1941.
53. Kim KS, Choi K, Park C, Hwanga H, Lee I: SUPPRESSOR OF
FRIGIDA4, encoding a C2H2-type zinc finger protein,
represses flowering by transcriptional activation of Arabidop-
sis FLOWERING LOCUS C. Plant Cell 2006, 18:2985-2998.
54. Folta KM, Staton M, Stewart PJ, Jung S, Bies DH,Jesudurai C, Main D:
Expressed sequence tags (ESTs) and simple sequence repeat
(SSR) markers from octoploid strawberry (Fragaria x anan-
assa). BMC Plant Biol 2005, 5:12.
55. Levy YY, Mesnage S, Mylne JS, Gendall AR, Dean C: Multiple roles
of Arabidopsis VRNI in vernalization and flowering time con-
trol. Science 297:243-246.
56. MylneJS, Barrett L, Tessadori F, Mesnage S, Johnson L, Bernatavichute
VN, Jacobsen SE, Fransz P, Dean C: LHP I, the Arabidopsis homo-
logue of HETEROCHROMATIN PROTEIN I, is required for
epigenetic silencing of FLC. PNAS 2006, 103:5012-5017.
57. Lee JH, Yoo SJ, Park SH, Hwang I, Lee JS, Ahn JH: Role of SVP in
the control of flowering time by ambient temperature in
Arabidopsis. Genes Dev 2007, 21:397-402.


58. Wagner D, Sablowski RWM, Meyerowitz EM: Transcriptional acti-
vation of APETALAI by LEAFY. Science 1999, 285:582-584.
59. Turck F, Fornara F, Coupland G: Regulation and identity of flori-
gen: FLOWERING LOCUS T moves central stage. Annu Rev
Plant Biol 2008, 59:573-594.
60. Zagotta MT, Hicks KA, Jacobs Cl, Young JC, Hangarter RP, Meeks-
Wagner D: The Arabidopsis ELF3 gene regulates vegetative
photomorphogenesis and the photoperiodic induction of
flowering. PlantJ 1996, 10:691-702.
61. Weebadde CK, Wang D, Finn CE, Lewers KS, LubyJJ, BushakraJ, Sju-
lin TM, HancockJF: Using a linkage mapping approach to iden-
tify QTL for day-neutrality in the octoploid strawberry. Plant
Breed 2008, 127:94-101.
62. Ahmadi H, Bringhurst RS, Voth V: Modes of inheritance of pho-
toperiodism in Fragaria. J Amer Soc Hort Sci 1990, I 15:146-452.
63. Shulaev V, Korban SS, Sosinski B, Abbott AG, Aldwinckle HS, Folta
KM, lezzoni A, Main D, Arus P, Dandekar AM, Lewers K, Brown SK,
Davis TM, Gardiner SE, Potter D, Veilleux RE: Multiple models for
Rosaceae genomics. Plant Physiol 2008, 147:985-1003.
64. Takada S, Goto K: Terminal flower 2, an Arabidopsis homolog
of heterochromatin protein I, counteracts the activation of
Flowering locus T by Constans in the vascular tissues of leaves
to regulate flowering time. Plant Cell 2003, 15:2856-2865.
65. Sawa M, Nusinow DA, Kay SA, Imaizumi T: FKF I and GIGANTEA
complex formation is required for day-length measurement
in Arabidopsis. Science 2007, 3 18:261-265.
66. Hecht V, Foucher F, Ferrandiz C, Macknight R, Navarro C, Morin J,
Vardy ME, Ellis N, Beltran J, Rameau C, Weller JL: Conservation of
Arabidopsis flowering genes in model legumes. Plant Physiol
2005, 137:1420-1434.
67. Alabadi D, Oyama T, Yanovsky MJ, Harmon FG, Mas P, Kay SA:
Reciprocal regulation between TOCI and LHYICCAI within
the Arabidopsis circadian clock. Science 2001, 293:880-883.
68. Bohlenius H: Control of flowering time and growth cessation
in Arabidopsis and Populus trees. In PhD thesis Swedish University
of Agricultural Sciences, Umea; 2008.
69. Wang R, Farrona S, Vincent C, Joecker A, Schoof H, Turck F, Alonso-
Blanco C, Coupland G, Albani MC: PEPI regulates perennial flow-
ering in Arabis alpina. Nature 2009, 459:423-427.
70. Zhang X, Clarenz 0, Cokus S, Bernatavichute YV, Pellegrini M,
Goodrich J, Jacobsen SE: Whole genome analysis of histone H3
lysine 27 trimethylation in Arabidopsis. PloS Biol 2007, 5:e 129.
71. Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T,
Baba T, Yamamoto K, Umehara Y, Nagamura Y, Sasaki T: Hdl, a
major photoperiod sensitivity quantitative trait locus in rice,
is closely related to the Arabidopsis flowering time gene
CONSTANS. Plant Cell 2000, 12:2473-2484.
72. Sargent DJ, Clarke J, Simpson DW, Tobutt KR, Arus P, Monfort A,
Vilanova S, Denoyes-Rothan B, Rousseau M, Folta KM, Bassil NV, Bat-
tey NH: An enhanced microsatellite map of diploid Fragaria.
Theor AppI Genet 2006, I 12:1349-1359.
73. Roberts AV, Blake PS, Lewis R, TaylorJM, Dunstan DI: The effect of
gibberellins on flowering in roses. J Plant Growth Regul 1999,
18:113-119.
74. Monte D, Somerville S: Pine tree method for isolation of plant
RNA. In DNA microarrays: a molecular cloning manual Edited by: Bow-
tell D, Sambrook J. New York: Cold Spring Harbour Laboratory
Press; 2002:124-126.
75. Laitinen RAE, Immanen J, Auvinen P, Rudd S, Alatalo E, Paulin L, Ain-
asoja M, Kotilainen M, Koskela S, Teeri TH, Elomaa P: Analysis of
the floral transcriptome uncovers new regulators of organ
determination and gene families related to flower organ dif-
ferentiation in Gerbera hybrida (Asteraceae). Genome Res
2005, 15:475-486.
76. Tuskan GA, DiFazio S, Jansson S, Bohlmann J, Grigoriev I, et al.: The
genome of black cottonwood, Populus trichocarpa (Torr. &
Gray). Science 2006, 313:1596-1604.
77. Childs KL, Hamilton JP, Zhu W, Ly E, Cheung F, Wu H, Rabinowicz
PD, Town CD, Buell CR, Chan AP: The TIGR plant transcript
assemblies database. Nucleic Acids Res 2007, 35:D846-D85 I.
78. Mockler T, Yang H, Yu X, Parikh D, Cheng Y, Dolan S, Lin C: Regu-
lation of photoperiodic flowering by Arabidopsis photore-
ceptors. PNAS 2003, 100:2140-2145.
79. Guo HW, Yang WY, Mockler TC, Lin CT: Regulation of flowering
time by Arabidopsis photoreceptors. Science 1998,
279:1360-1363.



Page 15 of 16
(page number not for citation purposes)


BMC Plant Biology 2009, 9:122








BMC Plant Biology 2009, 9:122


80. Kim W, Fujiwara S, Suh S, Kim J, Kim Y, Han L, David K, Putterill J,
Nam HG, Somers DE: ZEITLUPE is a circadian photoreceptor
stabilized by GIGANTEA in blue light. Nature 2007,
449:356-360.
81. Kim D, Kang J, Yang S, Chung K, Song P, Park C: A phytochrome-
associated protein phosphatase 2A modulates light signals in
flowering time control in Arabidopsis. Plant Cell 2002,
14:3043-3056.
82. Staiger D, Allenbach L, Salathia N, Fiechter V, Davis SJ, Millar AC,
Chory J, Fankhauser C: The Arabidopsis SRRI gene mediates
phyB signaling and is required for normal circadian clock
function. Genes Dev 2003, 17:256-268.
83. Hazen SP, Schultz TF, Pruneda-Paz JL, Borevitz JO, Ecker JR, Kay SA:
LUX ARRHYTHMO encodes a myb domain protein essen-
tial for circadian rhythms. PNAS 2005, 102:10387-10392.
84. Doyle MR, Davis SJ, Bestow RM, McWatters HG, Kozma-Bognar L,
Nagy F, Millar AJ, Amasino MR: The ELF4 gene controls circadian
rhythms and flowering time in Arabidopsis thaliana. Nature
2002, 419:74-77.
85. Nakamichi N, Kita M, Niinuma K, Ito S, Yamashino T, Mizoguchi T,
Mizuno T: Arabidopsis clock-associated pseudo-response regu-
lators PRR9, PRR7 and PRR5 coordinately and positively reg-
ulate flowering time through the canonical CONSTANS-
dependent photoperiodic pathway. Plant Cell Physiol 2007,
48:822-832.
86. Noh B, Lee S, Kim H, Yi G, Shin E, Lee M, Jung KMR, Doyle KMR,
Amasino RM, Noh Y: Divergent roles of a pair of homologous
jumonji/zinc-finger-class transcription factor proteins in the
regulation of Arabidopsis flowering time. Plant Cell
16:2601-2613.
87. Hanzawa Y, Money T, Bradley D: A single amino acid converts a
repressor to an activator of flowering. PNAS 2005,
102:7748-7753.
88. Chen M, Ni M: RFI2, a RING-domain zinc finger protein, neg-
atively regulates CONSTANS expression and photoperiodic
flowering. Plant] 2006, 46:823-833.
89. Cai X, BallifJ, Endo S, Davis E, Liang M, Chen D, DeWald D, Kreps J,
Zhu T, Wu Y: A putative CCAAT-binding transcription factor
is a regulator of flowering timing in Arabidopsis. Plant Physiol
2007, 145:98-105.
90. Pien S, Fleury DF, Mylne JS, Crevillen P, Inz6 D, Avramova Z, Dean C,
Grossniklaus U: ARABIDOPSIS THITHORAXI dynamically
regulates FLOWERING LOCUS C activation via histone 3
lysine 4 trimethylation. Plant Cell 2008, 20:580-588.
91. Zhang H, van Nocker S: The VERNALIZATION INDEPENDENCE
4 gene encodes a novel regulator of FLOWERING LOCUS C.
Plant] 2002, 3 1:663-673.
92. Gendall AR, Levy YY, Wilson A, Dean C: The VERNALIZATION 2
gene mediates the epigenetic regulation of vernalization in
Arabidopsis. Cell 2001, 107:525-535.
93. Chanvivattana Y, Bishopp A, Schubert D, Stock C, Moon Y, Sung ZR,
Goodrich J: Interaction of Polycomb-group proteins control-
ling flowering in Arabidopsis. Development 2004, 13 1:5263-5276.
94. MacKnight R, Bancroft I, Page T, Lister C, Schmidt R, Love K, West-
phal L, Murphy G, Sherson S, Cobbett C, Dean C: FCA, a gene con-
trolling flowering time in Arabidopsis thaliana encodes a
protein containing RNA binding domains. Cell 1997,
89:737-745.
95. Schomburg FM, Patton DA, Meinke DW, Amasino RM: FPA, a gene
involved in floral induction in Arabidopsis thaliana, encodes a
protein containing RNA-recognition motifs. Plant Cell 2001,
13:1427-1436.
96. Lim MH, Kim J, Kim YS, Chung KS, Seo YH, Lee I, Kim J, Hong CB,
Kim HJ, Park CM: A new Arabidopsis thaliana gene, FLK,
encodes a RNA binding protein with K homology motifs and
regulates flowering time via FLOWERING LOCUS C. Plant Cell
2004, 16:731-740.
97. Simpson GG, Dijkwel PP, Quesada V, Henderson I, Dean C: FY is a
RNA 3'end-processing factor that interacts with FCA to con-
trol the Arabidopsis thaliana floral transition. Cell 2003,
113:777-787.
98. Wang X, Zhang Y, Ma Q, Zhang Z, Xue Y, Bao S, Chong K: SKB I-
mediated symmetric dimethylation of histone H4R3 con-
trols flowering time in Arabidopsis. EMBOJ 2007, 26:1934-1941.
99. Lee I, Aukerman MJ, Gore SL, Lohman KN, Michaels SD, Weaver LM,
John MC, Feldmann KA, Amasino RM: Isolation of LUMINIDE-


http://www. biomedcentral.com/1471-2229/9/122


PENDENS: a gene involved in the control of flowering time in
Arabidopsis thaliana. Plant Cell 1994, 6:75-83.
100. Cheng H, Qin L, Lee S, Fu X, Richards DE, Cao D, Luo D, Harberd
NP, Peng J: Gibberellin regulates Arabidopsis floral develop-
ment via suppression of DELLA protein function. Development
2004, 13 1:1055-1064.
101. Tseng TS, Salome PA, McClung CR, Olszewski NE: SPINDLY and
GIGANTEA interact and act in Arabidopsis thaliana path-
ways involved in light responses, flowering and rhythms in
leaf movements. Plant Cell 2004, 16:1550-1563.
102. Magome H, Yamaguchi S, Hanada A, Kamiya Y, Oda K: Dwarf and
delayed-flowering I, a novel Arabidopsis mutant deficient in
gibberellin biosynthesis because of overexpression of puta-
tive AP2 transcription factor. Plant] 2004, 37:720-729.
103. Kania T, Russenberger D, Peng S, Apel K, Melzer S: FPF I promotes
flowering in Arabidopsis. Plant Cell 1997, 9:1327-1338.
104. Aukerman MJ, Sakai H: Regulation of flowering time and floral
organ identity by a microRNA and its APETALA2-like target
genes. Plant Cell 2003, 15:2730-274 1.


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