Biology Reference
In-Depth Information
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 Ge-
nome Institute [76] using cut-off value 1e-10. tBLASTx was performed against
TIGR plant transcript assemblies of Malus x domestica, Oryza sativa and Vitis
vinifera [77], and GDR Fragaria and Rosaceae Contigs using cut-off value 1e-
10. For MIPS BLAST hits corresponding functional classes and Gene Ontology
classes were obtained from Functional Classification Catalogue (Version 2.1) and
GO annotation for Arabidopsis thaliana (Version 1.1213).
Homologs of Arabidopsis flowering time genes were searched from GDR Fra-
garia contig and EST databases using tBLASTx algorithm and Arabidopsis protein
sequences as a query. Homologous sequences passing a cut-off value 1e-10 were
further analysed by BLASTx algorithm against Arabidopsis protein database, and
sequences showing highest sequence homology with the corresponding Arabidopsis
genes were selected. The sequences lacking from Fragaria were similarly searched
from GDR Rosaceae EST database and from Rosaceae protein database 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 18°C. During
germination, plants were illuminated using 400 W SON-T lamps (Airam) for 12
h daily (90 ± 10
µ
mol m
-2
s
-1
at plant height plus natural light) and incandescent
lamps were used for low-intensity daylength extension (5 ± 1
µ
mol 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°C), for five weeks. In LD, in-
candescent lamps were used for low-intensity daylength extension (5 ± 1
µ
mol 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 carried
out at the greenhouse using darkening curtains, while LD treatments (photope-
riod 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 treatments, 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 synthesized from total RNA using Superscript III RT kit
