Biology Reference
In-Depth Information
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 conditions,
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 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 genotypes, 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 [Genbank:GH202443-GH203412] and 1184
EB ESTs [GenBank:GH201259-GH202442] passed quality checking. Pairwise
comparison of these EST datasets revealed that there was very little overlap be-
tween the libraries. However, general distribution of the sequences to functional
categories (FunCat classification) did not reveal any major differences between
the two libraries.
BLASTx searches against Arabidopsis, Swissprot and non-redundant data-
bases 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
revealed highest number of hits with Populus trichocarpa (Table 1). We also
performed tBLASTx searches against TIGR plant transcript assemblies of
Malus × domestica, 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 efficiently enriched rare transcripts
in the libraries.
