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Molecular-Genetic Characterization of Flowing in Cultivated Strawberry
(Frageria xananassa)
Kevin Folta et al., 2005
Introduction
The 2005 project had two major objectives to be accomplished under the $2000 award.
The first was to identify the orthologs of photoperiod genes in Fragaria. The goal would
be met starting with sequence from the model system. Using a series of methods, namely
degenerate PCR and colony hybridization based on sequence from the model system, the
similar genes in Fragaria could be isolated and characterized.
The second part of the project aimed to study the expression of these genes in long and
short day plants to test if their expression patterns agreed with those observed in the
model systems.
Methods
Degenerate PCR proved to be an unproductive avenue. Many products were generated
and in some cases cloned and sequenced, yet revealed no products of interest. In
September of this year a new strategy was adopted. A series of cDNA libraries was
constructed in InVitrogen Gateway vectors. Unlike our previous library (Folta et al.,
2005) which was vector based, the new library was resident to E. coli and featured the
rapid downstream capacities of the Gateway system. Three libraries were generated. The
first was a library from RNA isolated from new floral buds, the second, a fruit-ripening
series, the third representing etiolated/light grown seedlings. All of these libraries were
probed with radiolabeled cDNA probes to identify homologs of Fragaria photoperiod
genes.
Gene expression studies were carried out in ‘Strawberry Festival’ (a short day plant),
‘Diamonte’ (a day neutral cultivar) and ‘Laboratory Festival #9’ a laboratory strain that
can be easily transformed and regenerated where photoperiod sensitivity has not been
established. Plants were grown under long days (16 h light, 8 h dark) or under short days
(8 h light, 16 h dark). RNA was harvested at 3 hour intervals, fractionated on denaturing
agarose gels and blotted to nylon membranes. No further analysis as been undertaken at
this point.
Results
1. The 3’ end of at least six CONSTANS clones has been obtained. Using a primer
conserved against all Co coding regions and a universal primer in a vector, a
series of PCR products were generated, cloned and sequenced. The varying
copies are consistent with the fact that the Co gene family is extensive in all cases
studied. Only the 3’ half has been obtained and matches well with the canonical
Constans gene as well as several Constans-like genes in Arabidopsis. Recently,
the Co region from Fragaria was used to screen the Gateway clones. No clones
were recovered from developing flowers, but 10 clones were identified in
2.
3.
4.
5.
6.
etiolated/early light-grown seedling ESTs. These clones are larger and likely
contain the complete sequence.
A full-length clone of the Fragaria SUPPRESSOR OF CONSTANS gene was
isolated and characterized. SOC is a critical component downstream of the floral
regulator CONSTANS. The sequence bears strong identity to the Arabidopsis
version. .
Sequence similar to Flowering Locus T has been obtained. FT, like SOC operates
downstream of CONSTANS to integrate the light signals to the meristem.
Several LEAFY constructs have been obtained, but none represent a bona fide
LEAFY ortholog. These trials are still underway.
Like LEAFY, several PHYTOCHROME sequences have been identified with
local homology to this major class of light sensors. However, none represents a
phytochrome in totality. The recent acquisition of phy sequence from Fragaria
vesca (kindly provided from Dr. Tom Davis) will allow screening colonies with
homologous sequences, possibly leading to identification of these genes.
HEME OXYGENASE 1 is required for synthesis of the phy chromophore. A fulllength ortholog has been identified.
Discussion
Since photoperiod is a critical factor in strawberry production, it is of great importance to
understand its molecular mechanism. Such description will help in the design of
molecular markers to aid cultivar improvement via traditional breeding, and their
characterization will permit development of transgenic plants. These plants may be
useful tools in the laboratory or possibly the field.
Study of the photoperiod pathway in strawberry is also important because strawberry has
some interesting intricacies that suggest it will deviate from the current models.
Arabidopsis and rice are long and short-day plants, respectively. Although they contain
the same photoperiod components (CO, SOC etc), yet they work in opposing ways.
However, strawberry maintains all of these same proteins in cultivars with greatly
varying photoperiod sensitivities, yet at the same time represent a remarkably narrow
genetic base. In this case, strawberry may be a significant reference species to dissect the
intricacies of photoperiodic flowering, as some slight deviation in genotype has a
significant effect on phenotype.
The results indicate that a number of alleles of critical floral regulators have been
identified in strawberry. Current efforts under this award will test to see if these
sequences may complement Arabidopsis mutants, and they also will be overexpressed in
the ‘Laboratory Festival #9’ background. The blots that have been prepared will be
probed to describe expression patterns to determine if Co, Soc and Ft expression are
consistent with what has been observed in the model systems.
The libraries generated also represent a substantial community resource. These will be
freely distributed upon publication, as will all clones characterized.
These activities represent substantial return on investment for NASGA, as an important
set of relevant regulatory genes has now been identified in Fragaria. The results will be
prepared for publication in Plant Physiology in 2006. The generous award from NASGA
will be appropriately acknowledged. All genes identified will be deposited to public
databases and the contributions of NASGA will be cited accordingly.