Progress 06/01/03 to 05/31/07
Outputs
A cDNA library was constructed from unopened flower buds of Fragaria vesca ssp. vesca Yellow Wonder. Average insert size was about 1.5 kb. Approximately 5,500 clones of 1.5 kb average insert size were picked. About 3,300 randomly selected clones were sequenced. Of these, 2717 high quality strawberry EST (expressed sequence tag) sequences were obtained, and have been deposited into the GenBank EST database, under accession numbers DV438013-DV440729. Bioinformatic analysis on this data set performed by the staff of the Genome Database for the Rosaceae (GDR) resulted in the identification of 1910 unique expressed sequence tag (EST) sequences (unigenes). This result substantially exceeds the stated project goal of obtaining 500 Fragaria unigene sequences. Only 1235 of these unigenes had Blast hits to Rosaceae family ESTs, indicating that 675 of these new Fragaria ESTs were novel within the existing Rosaceae EST database. About 150 of the Fragaria EST sequences contained
simple sequence repeat (SSR) loci with five or more motif repeats. These gene-based SSRs may be of value as mapping markers. Intensive and persistent efforts to construct a bacterial artificial chromosome (BAC) library from F. vesca ssp. americana var. Pawtuckaway were impeded by difficulties in obtaining sufficiently high molecular weight DNA (~150 kb) in sufficient quantities for library construction. As an alternative means of fulfilling the project goal of constructing large insert genomic library resources for Fragaria vesca, a fosmid library was constructed. Genomic DNA from var. Pawtuckaway was physically sheared and cloned into the Epicentre pCC1FOS vector, and approximately 33,000 clones of ~40 kb average insert size were picked and spotted in duplicate on sets of two library filters. Filters have been successfully probed with over 20 single copy gene probes, one gene family probe, and chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) probe sets. The combined cpDNA and
mtDNA clone content of the library was about 11%. After correction for organelle insert content, the nuclear genome coverage of the library is about 6x. Library filter sets have been made available to other investigators upon request. In partial fulfillment of objective 3, construction of an F. vesca physical map was initiated: selected fosmid clones have been fingerprinted, end-sequenced, and/or completely sequenced to high resolution. With support from a separate grant, over 1.75 Mbp of genomic sequence (encompassing 50 genomic samples of ~35 kb each, deposited as GenBank accession numbers EU024823-EU024872) has been obtained, providing a highly informative, initial glimpse into the structure and composition of the Fragaria genome. Conservation of localized micro-colinearity between Fragaria and Arabidopsis was observed at several sites. PCR amplification of over 20 gene pair intervals (consisting of two adjacent genes and their respective intergenic region) has provided evidence of
substantial conservation of micro-colinearity between Fragaria and other rosaceous species, including peach, raspberry, and rose.
Impacts
The diploid strawberry model species, Fragaria vesca, was employed in the development of new functional and structural genomics resources of high impact, advancing both basic knowledge about the strawberry genome and opportunities for practical application to strawberry varietal improvement. The floral cDNA library and ~2700 F. vesca EST sequences deposited in GenBank have been employed by other investigators in linkage mapping and gene expression studies. By enabling the generation of 1.75 Mbp of strawberry genomic sequence samples, the large insert genomic library we constructed has provided a launching point for numerous collaborations, both domestic and international, and has spurred new investigations in Rosaceae comparative genomics and transferable marker development. These genomic resources and initiatives provide a basis for connecting specific genes to specific traits in the octoploid, cultivated strawberry, paving the way for implementation of gene-based,
marker assisted selection as a tool for strawberry breeders. Opportunity for cross-species comparisons of gene sequence and composition, as well as genome organization and linkage group structure, between Fragaria and other members of the economically important Rosaceae family has been significantly enhanced, expanding the relevance of the project results to peach, cherry, apple, rose, brambles, and many other Rosaceous species. Three undergraduate and two graduate students and a post doc have received intensive training in plant genomics, contributing to the next generation of professionals in this field.
Publications
- Brese, RL. 2006. The development and utilization of EST (expressed sequence tag) resources in the diploid strawberry model system. M.S. Thesis. University of New Hampshire, Durham, NH 03824. 95 pp.
- Davis TM, Denoyes-Rothan B, and Lerceteau-Kohler E. 2007. Strawberry. In: Kole C (ed) Genome Mapping & Molecular Breeding in Plants IV: Fruits and Nuts. Springer, Heidelberg, Berlin, New York, Tokyo.
- Davis TM, Folta KM, Shields ME, and Zhang Q. 2007. Gene pair markers: an innovative tool for comparative linkage mapping. Proceedings, North American Strawberry Symposium. February 9-12, Ventura, California. (accepted)
- Shields, ME. 2005. Construction and characterization of a large-insert genomic library for Fragaria (Rosaceae). M.S. Thesis. University of New Hampshire, Durham, NH 03824. 141 pp.
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Progress 10/01/04 to 09/30/05
Outputs
About 3,300 randomly selected clones from a cDNA library constructed from unopened flower buds of Fragaria vesca ssp. vesca Yellow Wonder were sequenced. Of these, 2717 high quality strawberry EST (expressed sequence tag) sequences were obtained, and have been deposited into the GenBank EST database, under accession numbers DV438013-DV440729. Bioinformatic analysis on this data set performed by the staff of the Genome Database for the Rosaceae (GDR) resulted in the identification of 1910 unique expressed sequence tag (EST) sequences (unigenes). This result substantially exceeds the stated project goal of obtaining 500 Fragaria unigene sequences. Only 1235 of these unigenes had Blast hits to Rosaceae family ESTs, indicating that 675 of these new Fragaria ESTs are novel within the existing Rosaceae EST database. About 150 of the Fragaria EST sequences contained simple sequence repeat (SSR) loci with five or more motif repeats. These gene-based SSRs may be of value as
mapping markers. The Fragaria ESTs are being cross-referenced to mapped peach ESTs as a basis for selecting markers for comparative physical and linkage mapping. In fulfillment of the project goal of constructing large insert genomic library resources for Fragaria vesca, a fosmid library of about 33,000 clones was constructed from F. vesca ssp. americana Pawtuckaway. Library filters have been probed to recover fosmids containing over 15 genes of interest, including genes of the anthocyanin pathway (chalcone synthase, chalcone isomerase, anthocyanidin synthase, flavanone 3-hydroxylase), alcohol dehydrogenase, leafy, constans, phytochrome A, pistillata, four NBS-LRR resistance-like genes, and other genes of potential practical interest. All probes tested to date have resulted in the detection of fosmids carrying the targeted genes. A probe based on a peach (Prunus persica) transcription factor that exists as a multi-gene cluster in peach has been used to probe the strawberry fosmid
filters, and subsequent preliminary fosmid fingerprinting has tentatively identified a single contig consisting of 24 fosmid clones. Analysis of fosmid sequences obtained under separate funding has detected a considerable degree of microsynteny conservation between Fragaria and Arabidopsis.
Impacts
Further progress has been achieved in the use of the diploid model species, Fragaria vesca, to develop functional and structural genomics tools for strawberry. These genomic tools will provide a basis for connecting specific genes to specific traits in the octoploid, cultivated strawberry, paving the way for implementation of gene-based, marker assisted selection as a tool for strawberry breeders. Opportunity for cross-species comparisons of gene sequence and composition, as well as genome organization and linkage group structure, between Fragaria and other members of the economically important Rosaceae family has been significantly enhanced, expanding the relevance of the project results to peach, cherry, apple, rose, brambles, and many other Rosaceous species.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
A cDNA library was constructed from unopened flower buds of Fragaria vesca ssp. vesca Yellow Wonder. Average insert size was about 1.5 kb. Approximately 5,500 clones of 1.5 kb average insert size were picked, and sequencing has been initiated. Efforts to construct a bacterial artificial chromosome (BAC) library from F. vesca ssp. americana Pawtuckaway were impeded by difficulties in obtaining sufficiently high molecular weight DNA (150 kb) in sufficient quantities for library construction. Efforts are under way to overcome these difficulties. Meanwhile, a fosmid library has been constructed from this genotype. Genomic DNA was physically sheared and cloned into the Epicentre pCC1FOS vector, and approximately 33,000 clones of 40 kb average insert size were picked and spotted in duplicate on a set of two library filters. Filters have been successfully probed with three single copy gene probes, one gene family probe, and chloroplast DNA (cpDNA) and mitochondrial DNA
(mtDNA) probe sets. The combined cpDNA and mtDNA clone content of the library was about 11%. After correction for organelle insert content, the nuclear genome coverage of the library is about 6x. Library filter sets have been made available to other investigators upon request. As steps toward construction of an F. vesca physical map, selected fosmid clones have been fingerprinted, and selected gene probes have been used to probe the library to identify gene-based contigs.
Impacts
Substantial progress has been achieved in the use of the diploid model species, Fragaria vesca, to develop functional and structural genomics tools for strawberry. These genomic tools will provide a basis for connecting specific genes to specific traits in the octoploid, cultivated strawberry, paving the way for implementation of gene-based, marker assisted selection as a tool for strawberry breeders. Opportunity for cross-species comparisons of gene sequence and composition, as well as genome organization and linkage group structure, between Fragaria and other members of the economically important Rosaceae family has been significantly enhanced, expanding the relevance of the project results to peach, cherry, apple, rose, brambles, and many other Rosaceous species.
Publications
- No publications reported this period
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Progress 06/01/03 to 09/30/03
Outputs
Nuclei and high molecular weight DNA were isolated from young leaves of the diploid strawberry F. vesca var. Pawtuckaway for the purpose of genomic library construction. Total RNA and polyA mRNA were isolated from unopened flower buds of diploid strawberry F. vesca var. Yellow Wonder for the purpose of cDNA library construction.
Impacts
The successful isolation of high molecular weight DNA and polyA mRNA from the diploid strawberry, Fragaria vesca, is a necessary first step toward developing genomics resources in this species, a putative A genome donor to the octoploid, cultivated strawberry, F. x ananassa.
Publications
- No publications reported this period
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