Gene Pair Haplotypes and Sequence Samples from Strawberry (Rosaceae): Multi-Purpose, Transferable Resources for Genomics and Variety Improvement


NRI Award #2005-35300-15467
PI: Thomas M. Davis, Co-PI: Kevin M. Folta, Philip SanMiguel, Cooperator: Jeff Bennetzen
Department of Plant Biology, University of New Hampshire
Durham, NH 03824
E-mail: tom.davis@unh.edu
Telephone: 603-862-3217
Websites: www.strawberrygenes.org (TM Davis, under development)
http://arabidopsisthaliana.com/strawberry/ (KM Folta, active)


Accomplishments:

We have accomplished our project sequencing objective by sequencing 50 fosmid inserts from a genomic library of the strawberry diploid model species Fragaria vesca (ssp. americana var. Pawtuckaway). Based upon sequence assembly lengths, the fosmid inserts ranged in size from about 28 kb to 45 kb, with an average insert size of ~35 kb. A cumulative total of 1.75 Mb of genomic sequence was obtained (slightly under 1% of the 200 Mb F. vesca genome). Twenty of the 50 sequenced fosmid clones were selected via library probing, while 30 were selected at random (after pre-screening to exclude organellar inserts). The targeted clones were selected using probes for the following genes: (flowering-related) CONSTANS, Suppressor of CONSTANS, Leafy, PhyA, Hy5, Pistillata, Apetala3; (fruit color and flavor) Chalcone Synthase (CHS), Chalcone Isomerase (CHI), Dihydrofolate Reductase (DFR), Regulator of Anthocyanin Synthesis (RAN), Linalool Synthase (two clones); (other metabolic) Alcohol Dehydrogenase (ADH), Granule-Bound Starch Synthase I (GBSSI); (disease resistance-like) gRGA1, gRGA2, gRGA3, cRGA1, Ve-like.

Sequence annotation has revealed the following. Gene density is about 1 protein-encoding gene per 5-to-6 kb among the 20 gene-targeted fosmids, while it is somewhat lower and more variable among the randomly chosen fosmids. Intergenic (gene-to-gene) distances average about 3-4 kb. Several instances of tandemly duplicated protein-encoding genes were encountered, including five of the 20 targeted candidate genes: ADH, CHS, Pistillata, Linalool Synthase, cRGA1. Identified repetitive sequences include a ribosomal RNA gene cluster, 300 SSRs (of at least five repeat units) distributed at a density of about 1 per 6 kb, at least one transposable element family, and a family of short repetitive sequences that is conserved between Fragaria and Rosa.

We have accomplished our project objectives in relation to developing and mapping novel “gene pair” markers in the diploid strawberry. Here, the term "gene pair" refers to two immediately adjacent genes. The gene pair marker concept is a novel development and important deliverable of the present grant. Based on our genomic (fosmid) sequences, we have developed 25 PCR primer pairs that amplify gene pair intervals distributed across the strawberry genome. The citing of a "forward" PCR primer in a proximal exon in one member of the gene pair, and the "reverse" primer in a proximal exon of the second member of the pair, allows amplification of the "gene pair interval", which includes the polymorphism-rich intergenic region as well as any exon (and intron) sequences flanked by the PCR primers. Using restriction enzyme digestion of gene pair PCR products to reveal polymorphisms, the linkage map positions of the 20 gene-targeted fosmid clones have been determined on the diploid F. vesca reference map, and mapping of the remaining clones is in progress. At least two clones have been mapped to each of the seven F. vesca linkage groups. These mapped clones will provide useful anchors for physical mapping.

Using DNA samples from various rosaceous species provided by thirteen collaborating investigators, we have begun to assess gene pair marker transferability in the Rosaceae. An initially developed gene pair locus based on the tandemly duplicated CHS gene can be amplified in strawberry, cherry, peach, rose, and Rubus, but was problematic in apple. A second gene pair locus, provisionally termed CDPK-BHLH, has been amplified in strawberry, cherry, rose, and Rubus, but not apple. The latter marker, which is located on F. vesca linkage group I, has been located on Prunus linkage group VII using the Prunus TxE bin mapping set. We anticipate that gene pair sites can be established from each fosmid sequence, providing the potential for development of up to 50 gene pair anchor loci for comparative mapping among rosaceous species.

Addressing our Gene Pair Haplotype (GPH) objective, an initial gene pair locus in fosmid clone 19M24 has been examined in depth in Fragaria. Three distinct haplotypes have been defined at this locus in octoploid Fragaria species, and results indicate that it will be possible to differentiate multiple haplotype forms in individual octoploid varieties, supporting the expectation that gene pair haplotypes can be used as an effective mapping tool in the octoploid, cultivated strawberry. Five additional gene pairs have been amplified and analyzed from the octoploid cultivar Strawberry Festival. Several of these are related to genes associated with photoperiodic flowering and constitute the basis for functional studies around the trait. The variability identified within and between genomes is being compared to diploid species to test subgenome origin and provide a basis for mapping in the octoploid species. Surprising results indicate more intricate and varied evidence of strawberry’s reticulate ancestry than first predicted, as the molecular data do not easily fit with classical cytological models. Ongoing work will bring additional resolution to this important question. To aid in GPH mapping in octoploid Fragaria, an octoploid F. virginiana hybrid (LB48) was crossed as female with F. vesca to create a unique mapping population consisting of ~60 pentaploid strawberry plants, and the mapping of GPH loci in this population has been initiated.

The work has demanded improvement of available DNA extraction protocols. Optimization and modification of existing protocols generated new means of isolating materials suitable for most downstream applications, with greater yield and quality. Additionally, a simple yet powerful gene-pair analysis software suite (“Gene Pair Detective”) has been developed. The program predicts potential colinearity between an unknown genome (in this case Fragaria) and a known genome (Arabidopsis, Populus) using EST sequences from any unknown genome.

Broad Impacts:

This project has leveraged the complementary strengths and backgrounds of the Co-PIs (Tom Davis and Kevin Folta) and cooperator (Jeff Bennetzen) in an integrated effort to advance the state of Fragaria and Rosaceae genomics and the understanding of plant genome composition in general. The resulting genomics resources will be of both immediate and long term value relevant to breeding and marker assisted selection for strawberry variety improvement. A welcome and only partially anticipated outcome of the project is the extent to which genomic resources developed for strawberry – specifically, genomic DNA sequence data and concomitant development of novel "gene pair markers" - will provide a comparative mapping resource that is proving highly transferable to other rosaceous species. The potential appeal of this resource has attracted cooperators from six different countries representing almost all of the major Rosaceae crop species, thereby strengthening Rosaceae community interaction and commonality of interest and effort to the benefit of all.

The pending release of the Gene Pair Detective program will speed further predictions of possible relationships between species and allow a basis for wet-lab testing for gene-pair variability. The program is applicable to any undersequenced species, allowing rapid elucidation of genomic sequence that may be used in a variety of applications of broad community relevance.

Deliverables
  • Publications
  • Folta, K.M., and T.M. Davis. 2006. Strawberry genes and genomes. Crit. Rev. Plant Sci. 25:399- 415.
  • Davis TM, Folta KM, Shields ME, and Zhang Q. 2007. Gene pair markers: an innovative tool for comparative linkage mapping. Proceedings of the 2007 North American Strawberry Symposium, ASHS Press, Alexandria, VA, in press.
  • Sargent DJ, Davis TM, Simpson DW. Rosaceae Crop Species Genomics – Fragaria. A: Structural genomics. In: Gardiner S and Folta KM (eds) Genetics and Genomics of Rosaceae. Springer, Heidelberg, Berlin, New York (anticipated publication Spring, 2008).
  • Presentations & Meeting Abstracts
  • Poulsen E, and Davis TM. 2005. Genome composition and evolution in Fragaria. UNH Undergraduate Research Conference. April 30, 2005. Poster # 24.
  • Davis TM, Folta KM, Shields ME, Brese RL, DiMeglio LM, and Zhang Q. 2005. New Genomics Resources for Strawberry. American Society for Horticultural Science, Annual Conference. Submitted talk.
  • Davis TM. Folta KM, Bennetzen J, and SanMiguel P. 2005. Gene Pair Haplotypes and Sequence Samples in Strawberry. American Society for Horticultural Science, Annual Conference. Invited talk.
  • Tombolato, D., Stewart, P.J., Davis, T.M., Folta, K.M. 2005. Gene pair haplotypes: Novel molecular markers for genetic studies in octoploid strawberry. University of Florida Plant Molecular and Cellular Biology Workshop, Daytona Beach, FL.
  • Tombolato, D.C.M., Stewart, P.J., Davis, T.M., Folta, K.M. 2005. Gene-pair haplotypes: novel, complex markers for linkage mapping in octoploid strawberry. Genetics Institute Symposium, University of Florida.
  • Davis TM, Shields ME, Zhang Q, Bennetzen JL, Pontaroli AC, SanMiguel P, Folta KM, Tombolato DCM. 2006. Sequence samples from the diploid strawberry model species, Fragaria vesca.). International Plant & Animal Genome Conference XIV, January 14-18, San Diego, CA. Poster presenter.
  • Davis TM, Folta KM, Bennetzen JL, SanMiguel P, Slovin J, Rabinowicz PD, Hancock JF, Lewers KS, van Nocker S, Wang D, Ashman T-L, Main D, Staton M. 2006. Functional and structural genomic resource development, and QTL mapping of day-neutrality and sexdetermination, in strawberry (Fragaria). International Plant & Animal Genome Conference XIV, January 14-18, San Diego, CA. Fruit and Nut Workshop: Invited speaker.
  • Davis TM. 2006. Sequence Samples and Gene Pair Haplotypes in Strawberry. 3rd International Rosaceae Genomics Conference, Napier, New Zealand, March 19-22. Workshop session speaker.
  • Tomolato, D.C.M., Stewart, P.J., Davis, T.M. and Folta, K.M. 2006. Gene-pair-haplotypes: Novel, complex markers for linkage mapping in diploid and octoploid strawberries. 17th Annual PMCB Workshop, Jacksonville Beach, FL.
  • Davis TM. 2007. Genomic Tools and Resources for strawberry (Fragaria). International Plant & Animal Genome Conference XV, January 12-17, San Diego, CA. Rosaceae Project Directors Meeting.
  • Davis TM, Shields ME, Zhang Q, Tombolato D, and Folta KM. 2007. Gene pair markers: An innovative tool for comparative linkage mapping in the Rosaceae family and in other taxa with small genomes. International Plant & Animal Genome Conference XV, January 12-17, San Diego, CA. Poster #193.
  • Davis TM. 2007. Gene pair markers: An innovative tool for comparative linkage mapping. North American Strawberry Symposium. Feb 9-12, Ventura, CA. Invited talk.
  • Folta, K.M. Structural, functional and translational genomics in strawberry. Washington State University, Pullman, WA. Invited talk.
  • Folta, K.M. 2007 The molecular control of day neutrality in cultivated strawberry. East Malling Research, Kent UK. Invited talk.
  • Web sites
  • www.strawberrygenes.org (UNH project website)
  • http://arabidopsisthaliana.com/strawberry/ (U. Florida project website)
  • Community resources generated (sequences, populations, plant materials, etc)
  • F. vesca genome library, ~33,000 clones, and filter sets.
  • Genomic sequences from fosmid inserts (~35 kb average size): GenBank numbers EU024823-EU024872
  • Pentaploid mapping population. This population of ~60 pentaploid strawberry plants was created via sexual hybridization between diploid F. vesca and an octoploid F. virginiana hybrid (LB48). The population is available to interested researchers.
  • The gene pair marker and gene pair haplotype concepts, and gene pair primer sets.
  • Gene Pair Detective computer program.
  • Training
  • Undergraduate
    • Poulsen E. 2005. Genome composition and evolution in Fragaria. UNH Undergraduate Research Conference. April 30, 2005. Poster # 24. Supported by SURF award, 2004. (Identification of diploid ancestors, relevant to genomic resource development.)
    • Orcheski, Ben. The genetic determination of sex expression in strawberry. Supported by UROP award, 2007. Senior Honors Thesis. UNH Undergraduate Research Conference. April 28, 2007. (Isolation of candidate sequences used as library probes.)
    • Jones, Mark. Analysis of intergenic variation in genes relevant to agriculturally-important traits in cultivated strawberry (F. ×ananassa)
  • Graduate
    • Poulsen, Elizabeth. Genome composition of allopolyploid strawberry species. Genetics M.S. Program. University of New Hampshire. In progress. (This project’s cope includes genotyping of GPH loci in a pentaploid strawberry mapping population.)
    • Orcheski, Benjamin. Genetics of sex determination in Fragaria. M.S. Program, Genetics. University of New Hampshire. In progress. (This project seeks to identify associations between gender (female versus hermaphrodite) and sex-determination candidate genes, the genomic sequences of which were obtained via sequencing of F. vesca fosmid clones.)
    • Tombolato, DCM. Structural genomics of Fragaria spp.-.Diploid and octoploid strawberries. Ph.D. Thesis. University of Florida, Gainesville FL (graduated August 2007).
    • Gonzalez, WF. Development of molecular markers for disease resistance in wild accessions of octoploid strawberry (F. ×ananassa). M.S. Thesis. University of Florida, Gainesville, FL. (withdrew from graduate school).
    • Stewart, PJ. The Fragaria CONSTANS gene family and the regulation of photoperiodic flowering. Ph.D. Thesis. University of Florida, Gainesville FL (graduated May 2007).
    • Mishra, V. Gene Pair Detective- Computationally assisted capture of intergenic sequence from poorly represented genomes. M.S. Thesis, University of Florida, Gainesville, FL (May, 2009; anticipated)
    • Melissa Hamner, University of Florida, Plant Molecular and Cellular Biology Program rotation student, assisted with gene pair marker mapping.
  • Post Doctoral
    • Zhang, Qian. Postdoctoral Research Associate, UNH. Fosmid library probing, fosmid handling, sequencing, genotyping, sequence analysis and annotation.
    • Pontaroli, Ana. Post doctoral training, Univ. of Georgia. Subcloning of Fragaria fosmids, sequence assembly, annotation and analysis.
    • Zhang, Kaichun. Visiting Scientist, Vice Director of Beijing Institute of Pomology and Forestry. Gene Pair Haplotypes from cultivated strawberry inform the origins of its diploid ancestry.
Collaborations:
  • Janet Slovin, USDA, Beltsville. Utilization of EST and genome library resources.
  • Dorrie Main, Genome Database for Rosaceae, Clemson/Washington State University.
  • Kim Lewers, USDA, Beltsville. Mapping and marker development.
  • Kim Hummer, USDA-NCGR, Corvallis. Fragaria germplasm development.
  • Nahla Bassil, USDA-NCGR, Corvallis. Germplasm evaluation and marker development.
  • Vladimir Shulaev, Virginia Bioinformatics Institute. EST sequencing, other.
  • Dan Sargent, East Malling Research, UK. Marker development, mapping.
  • Bert Abbott, Jahn Davik, Clemson. Strawberry-peach comparative genomics.
  • Sue Gardiner, HortReseach, NZ. Gene pair marker testing.
  • Pere Arus, Amparo Monfort, Werner Howad, IRTA, Spain. Mapping.
  • Jim Hancock, Michigan State University. Mapping.
  • Tia-Lynn Ashman, University of Pittsburgh. Anticipated candidate gene testing.
  • Timo Hytonen, University of Helsinki. Functional analysis of genes associated with photoperiodic flowering.
  • Fumi Takeda, USDA Kearneysville. Functional analysis of genes associated with photoperiodic flowering.
  • Natalia Peres, GCREC, University of Florida. Development of molecular markers for disease resistance in wild strawberry accessions.
  • Bobby Phills, Florida A&M, Translation of strawberry markers to red raspberry.
Contributors of Rosaceae DNA samples to Davis lab for use in assessing gene pair marker transferability: 13 investigators, six countries.
  • Irene Tierney, Scottish Crop Research Institute (Rubus)
  • Dan Sargent, East Malling Research, UK (Fragaria)
  • Amy Iezzoni, Michigan State University (Prunus - cherry)
  • Thomas Debener, University of Hannover, Germany (Rosa)
  • Elisabeth Dirlewanger, INRA, France (Prunus - cherry, peach)
  • Gennaro Fazio, Cornell, Geneva, NY (Malus - rootstock)
  • Schuyler Korban, University of Illinois at Urbana-Champagne (Malus)
  • Fabrice Faucher, INRA, France (Rosa)
  • Cameron Peace, Washington State University (Prunus - peach)
  • Kim Lewers, USDA Fruit Lab, Beltsville (Rubus)
  • Werner Howad/Pere Arus, IRTA, Spain (Prunus - peach, almond)
  • Patrick Lambert, INRA, France (Prunus)
  • David Byrne, Texas A&M University (Rosa)


A Diploid Platform for Strawberry Genomics POSTER #: 17


Accomplishments:

In fulfillment of project Objective 1, a cDNA library constructed using RNA from unopened flower buds of Fragaria vesca ssp. vesca ‘Yellow Wonder’ was used to generate an EST unigene set. Approximately 5,500 clones of 1.5 kb average insert size were picked, and about 3,300 randomly selected clones were sequenced. Of these, 2717 high quality EST (expressed sequence tag) sequences were obtained, and have been deposited into the GenBank EST database, under accession numbers DV438013-DV440734. 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 objective of 500 unigenes. Approximately 30% of these unigenes had no Blastn hits to Rosaceae family ESTs (as of 12/03/07), indicating that a substantial number of these Fragaria ESTs remain novel within the existing Rosaceae EST database and validating the initial expectation that flower buds would be a rich source of expressed sequence diversity. About 150 of the 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.

In relation to project Objective 2, after efforts to construct a bacterial artificial chromosome (BAC) library from F. vesca ssp. americana ‘Pawtuckaway’ were impeded by difficulties in obtaining sufficient quantities of high molecular weight DNA (~150 kb), a ‘Pawtuckaway’ fosmid library was constructed. 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 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 (physical mapping), selected fosmid clones have been fingerprinted, partially 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 was seen between Fragaria and model species including Arabidopsis, Populus, and Medicago, and evidence was obtained of substantial conservation of micro-colinearity between Fragaria and other rosaceous species, including peach, raspberry, and rose.

Broad 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, and by us in genomic sequence annotation. 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 crossspecies 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.

Deliverables
  • 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. 2006. Strawberry. In: Kole C (ed) Genome Mapping & Molecular Breeding in Plants IV: Fruits and Nuts. Springer, Heidelberg, Berlin, New York.
  • 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.
  • Davis TM, Folta KM, Shields ME, and Zhang Q. 2007. Gene pair markers: an innovative tool for comparative linkage mapping. Proceedings of the 2007 North American Strawberry Symposium, ASHS Press, Alexandria, VA, in press.
  • Community resources generated (sequences, populations, plant materials, etc)
  • F. vesca genomic library of ~33,000 clones in a fosmid vector. Library filter sets have been shared with cooperators (J. Slovin, B. Abbott). Clones have been provided to Bert Abbott (Clemson University) and the DOE-Joint Genomics Institute (JGI, Walnut creek, CA) for potential sequencing.
  • F. vesca cDNA library constructed from flower buds. Library clones shared with cooperators (V. Shulaev, Virginia Bioinformatics Institute; Timo Hytonen, University of Helsinki). ~2700 F. vesca EST sequences deposited in GenBank under accession numbers DV438013- DV440734.
  • Diploid (F. vesca) mapping population: F2 generation (n = 113) of the cross ‘Yellow Wonder’ x ‘Pawtuckaway’. This population is being used to construct a second generation F. vesca linkage map, and has been shared with cooperators (Dan Sargent, HRI, East Malling, UK; Pere Arus, IRTA, Cabrils, Spain).
  • Training
  • Undergraduate
    • deHaan, Kevin. 2004. Optimizing use of PCR to examine allelic diversity in octoploid strawberries. UNH Undergraduate Research Conference. May 1, 2004. Poster. Supported by SURF award, 2003.deHaan, Kevin. 2005. Absence of association between the ent-kaurene oxidase gene and the runnerless trait in Fragaria vesca. Senior Thesis. UNH Undergraduate Research Conference. April 30, 2005. Supported by SURF award, Spring 2004.
    • Poulsen E. 2005. Genome composition and evolution in Fragaria. UNH Undergraduate Research Conference. April 30, 2005. Poster # 24. Supported by SURF award, 2004. (Identification of diploid ancestors, relevant to genomic resource development.)
    • Danton, Ben. 2006. Witnessing evolution: cellular organelle and nuclear genomic incompatibilities as a source of reproductive isolation and divergence of species. Supported by SURF award, 2006. (Annotation of cDNA sequences.)
  • Graduate
    • Shields, Melanie. 2005. Construction and characterization of a large-insert genomic library for Fragaria (Rosaceae). Master of Science. Dept. of Plant Biology. University of New Hampshire. (Constructed and characterized F. vesca fosmid library.)
    • Brese, Robin. 2006. The development and utilization of EST (Expressed Sequence Tag) resources in the diploid strawberry model system. Master of Science. Dept. of Plant Biology. University of New Hampshire. (Constructed F. vesca cDNA library, bioinformatics.)
  • Post Doctoral
    • Zhang, Qian. Postdoctoral Research Associate, UNH. Fosmid library probing, fosmid handling, sequencing, genotyping, sequence analysis and annotation.
Collaborations:
  • Kevin Folta, University of Florida. Strawberry structural and functional genomics
  • Meg Staton, Clemson University.
  • Dorrie Main, Genome Database for Rosaceae, Clemson/Washington State University.
  • Janet Slovin, USDA, Beltsville. Utilization of EST and genome library resources.
  • Kim Lewers, USDA, Beltsville. Mapping and marker development.
  • Kim Hummer, USDA-NCGR, Corvallis. Fragaria germplasm development.
  • Dan Sargent, East Malling Research, UK. Marker development, mapping.
  • Bert Abbott, Jahn Davik, Clemson. Strawberry-peach comparative genomics.
  • Pere Arus, Amparo Monfort, IRTA, Spain. Marker development, mapping.
  • Timo Hytonen, University of Helsinki. Functional analysis of genes associated with photoperiodic flowering.