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 etiolated/early light-grown seedling ESTs. These clones are larger and likely contain the complete sequence.
  2. 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. .
  3. Sequence similar to Flowering Locus T has been obtained. FT, like SOC operates downstream of CONSTANS to integrate the light signals to the meristem.
  4. Several LEAFY constructs have been obtained, but none represent a bona fide LEAFY ortholog. These trials are still underway.
  5. 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.
  6. HEME OXYGENASE 1is required for synthesis of the phy chromophore. A full-length 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.