Browsing by Author "Shulaev, Vladimir"

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    Characterization of Polymorphic Microsatellites in Strawberry and Their Transferability to Other Genera in the Rosaceae Family
    Arora, Vishal (Virginia Tech, 2006-02-09)
    We investigated the transferability of 20 Fragaria vesca microsatellite primer pairs to 13 Fragaria vesca accessions, six Fragaria species and ten commercially important species in Rosaceae. Genetic diversity studies were carried among 16 diploid Fragaria accessions using these polymorphic microsatellites. The average number of alleles amplified for a polymorphic locus was 4.7 with maximum being 8.0 and minimum being 3.0. Observed heterozygosity ranged from 0.00 to 0.84 with an average of 0.28. Expected heterozygosity ranged from 0.33 to 0.91 with an average of 0.76. Power of discrimination varied from 0.43 to 0.92 with an average of 0.78. Transferability of microsatellites to F. orientalis (4x) and F. Ã ananassa (8x) was high, i.e., 18 (90%) primers produced amplicons. Cross species amplification within Rosaceae using these primers showed limited transference. Four microsatellites showed amplification for different species in Rosaceae. Products generated by UDF-003 and UDF-018 primers were sequenced. Sequencing results for UDF-018 showed that three species, i.e., Pyrus calleryana, Prunus persica and Rubus idaeus contained the expected microsatellite whereas another four, i.e., Cotoneaster salicifolius, Rosa rugosa, Amelanchier arborea and Potentilla fruticosa had conserved regions resulting in generation of amplicons. For UDF 003, Spirea xbumalda and Prunus persica did not contain a microsatellite although there was some sequence similarity with Fragaria. Size homoplasy, i.e., alleles of identical size with different numbers of repeats within the SSR was observed among Fragaria and Rosaceae species for primer UDF-018, suggesting a need for caution when interpreting SSR variation from band migration in the absence of DNA sequences.
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    Characterization of T-DNA integration sites within a population of insertional mutants of the diploid strawberry Fragaria vesca L.
    Ruiz-Rojas, Juan Jairo (Virginia Tech, 2010-11-01)
    Cultivated strawberry (Fragaria × ananassa) is an octoploid (2n=8x=56) species that belongs to the Rosaceae family and the high ploidy level makes genetic and molecular studies difficult. However, its commercial success because of its unique flavor and nutritious qualities has increased interest in the development of genomic resources. Fragaria vesca L. is a diploid (2n=2x=14) species with a small genome size (206 Mbp), short reproductive cycle, and facile vegetative and seed propagation that make it an attractive model for genomic studies. The availability of an efficient transformation methodology for Fragaria vesca has facilitated the use of a T-DNA mutagenesis system to develop a collection of several hundred insertional T-DNA mutants at Virginia Tech, using either of two commercially available vectors, pCAMBIA 1302 and 1304. In this study, we have used expression of the green fluorescent protein (GFP) as a tool to identify homozygous mutant lines. Three different approaches were conducted, first we identified 11 homozygous lines by PCR, then another 55 homozygous lines by absence of segregation of GFP expression in T2 seedlings, and finally we attempted to distinguish homozygous from hemizygous lines by relative GFP expression measured using a commercially available GFP meter. The latter methodology was unsuccessful due to uncontrolled variability in the readings. Continuing the characterization of our mutant population, we used thermal asymmetric interlaced PCR (TAIL-PCR) to obtain the nucleotide sequence of the genomic DNA regions that flank the T-DNA insertion sites in independent transgenic strawberry lines. Primers were designed that would amplify the derived strawberry flanking sequences in the two parents of an interspecific mapping population between the two diploid species, F. vesca x F. bucharica. The amplified products were sequenced and examined for the occurrence of SNPs (single nucleotide polymorphisms). The same primers were then used on the F2 mapping population. Segregation of SNP markers with previously mapped genetic markers allowed us to position 74 SNP markers, and hence their corresponding insertional mutants, on a well-populated genetic linkage map for the diploid strawberry. Finally, we analyzed the insertion site from more than 190 mutants looking at both the right and left borders of the T-DNA where microsimilarities of a few base pairs between ends of T-DNA and genomic DNA were observed, indicating that T-DNA integration had not occurred randomly in strawberry. We have also characterized the insertion sites through gene annotation found in the strawberry genome database.
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    Functional genomics through metabolite profiling and gene expression analysis in Arabidopsis thaliana
    Cortes Bermudez, Diego Fernando (Virginia Tech, 2008-07-25)
    In the post-genomic era, one of the most important goals for the community of plant biologists is to take full advantage of the knowledge generated by the Arabidopsis thaliana genome project, and to employ state-of-the-art functional genomics techniques to assign function to each gene. This will be achieved through a complete understanding of what all cellular components do, and how they interact with one another to produce a phenotype. Among the proteins encoded by the Arabidopsis genome are 24 related carboxyl methyltransferases that belong to the SABATH family. Several of the SABATH methyltransferases convert plant hormones, like jasmonic acid, indole-3-acetic acid, salicylic acid, gibberellins, and other plant constituents into methyl esters, thereby regulating the biological activity of these molecules and, consequently, myriad important physiological processes. Our research aims to decipher the function of proteins belonging to the SABATH family by applying a combination of genomics tools, including genome-wide expression analysis and gas-chromatography coupled with mass spectrometry-based metabolite profiling. Our results, combined with available biochemical information, provide a better understanding of the physiological role of SABATH methyltransferases, further insights into secondary plant metabolism and deeper knowledge of the consequences of modulating the expression of SABATH methyltransferases, both at the genome-wide expression and metabolite levels.
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    Genetic studies of phenotypic variants in the woodland strawberry, (Fragaria vesca)
    Holt, Sarah Hudson (Virginia Tech, 2011-09-02)
    The diploid woodland strawberry (Fragaria vesca) is a rapidly developing translational model for members of the family Rosaceae and other plants. This thesis represents some of the first forward genetics studies evaluating putative T-DNA insertional mutants in F. vesca. The observed phenotypes include alterations to floral development, anthocyanin pigmentation and leaf structure. The floral development mutant named green petal (gp) was not associated with the T-DNA insertions present. Based on similar phenotypes induced by mutation of transcription factors involved in floral development of Arabidopsis thaliana, we used a BLAST search of the F. vesca genome hybrid gene models to identify 30 candidate genes that may have caused the gp phenotype. Expression analysis of these genes revealed that it was due to a 37 bp deletion in a SEPALLATA3-like E-Class MADS box transcription factor. This mutation altered organ structure in the three inner whorls of the flower, affecting fertility and fruit development. The deletion was demonstrated to segregate with the mutant phenotype in a segregating population of 92 individuals, 22 of which had green petals. The anthocyanin biosynthesis mutant named white runner (wr) lacked red pigmentation in the stems and runners. The T-DNA insertion in this line was located in a highly repetitive LTR retrotransposon region, which complicated analysis. Segregation analysis of the wr lines revealed that the phenotype was unassociated with the T-DNA insertion as well. We used a targeted expression analysis of three critical structural genes in the flavonoid biosynthesis pathway that revealed a 20 bp deletion in the gene encoding flavanone 3-hydroxylase, an enzyme necessary for the production of flavonols, anthocyanins and proanthocyanidins. In an F2 segregating population, this deletion co-segregated with the phenotype. The third mutant line presented here displayed a curly leaf (cl) phenotype and was found to harbor a T-DNA insertion in a gene encoding a putative erythroblast macrophage attacher protein (EMP). Sequence and protein domain analysis indicated that FvEMP was related to the mammalian EMP protein that functions in cytoskeletal dynamics and red blood cell enucleation. Complementation analysis confirmed that introduction of the wild type FvEMP gene into the cl mutant plants restored wild type leaf phenotype. Further morphological analysis revealed additional pleiotropic effects of the mutation, including abnormalities in seed set and germination, pollen tube growth, adhesion of the abaxial epidermal layer to the mesophyll layer and reduced petiolule length. These phenotypes are consistent with actin binding and microtubule associated protein mutants in other plant species. Insertional mutagenesis is a critical molecular tool for model crop development. These studies highlight the precautions that must be taken when evaluating insertional mutants. These mutants are excellent tools for studying their respective disrupted gene function. The in depth molecular analysis of the mutants presented in this work was only possible because of the availability of the Fragaria vesca genome which was used extensively to identify T-DNA insertion sites and recover candidate gene sequences for expression analysis.
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    The Genome-Wide Early Temporal Response of Saccharomyces cerevisiae to Oxidative Stress Induced by Cumene Hydroperoxide
    Sha, Wei; Martins, Ana M.; Laubenbacher, Reinhard C.; Mendes, Pedro; Shulaev, Vladimir (PLOS, 2013-09-20)
    Oxidative stress is a well-known biological process that occurs in all respiring cells and is involved in pathophysiological processes such as aging and apoptosis. Oxidative stress agents include peroxides such as hydrogen peroxide, cumene hydroperoxide, and linoleic acid hydroperoxide, the thiol oxidant diamide, and menadione, a generator of superoxide, amongst others. The present study analyzed the early temporal genome-wide transcriptional response of Saccharomyces cerevisiae to oxidative stress induced by the aromatic peroxide cumene hydroperoxide. The accurate dataset obtained, supported by the use of temporal controls, biological replicates and well controlled growth conditions, provided a detailed picture of the early dynamics of the process. We identified a set of genes previously not implicated in the oxidative stress response, including several transcriptional regulators showing a fast transient response, suggesting a coordinated process in the transcriptional reprogramming. We discuss the role of the glutathione, thioredoxin and reactive oxygen species-removing systems, the proteasome and the pentose phosphate pathway. A data-driven clustering of the expression patterns identified one specific cluster that mostly consisted of genes known to be regulated by the Yap1p and Skn7p transcription factors, emphasizing their mediator role in the transcriptional response to oxidants. Comparison of our results with data reported for hydrogen peroxide identified 664 genes that specifically respond to cumene hydroperoxide, suggesting distinct transcriptional responses to these two peroxides. Genes up-regulated only by cumene hydroperoxide are mainly related to the cell membrane and cell wall, and proteolysis process, while those down-regulated only by this aromatic peroxide are involved in mitochondrial function.
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    Glutathione Dynamics in Arabidopsis Seed Development and Germination
    Sumugat, Mae Rose S. (Virginia Tech, 2004-12-09)
    Seed desiccation and germination have great potential for oxidative stress. Glutathione, one of the most abundant antioxidants in plant cells, is a crucial to the plant's defense mechanisms. To better understand glutathione's responses during these two stages, we examined its dynamics in wildtype Arabidopsis seeds and in a transgenic line containing an antisense glutathione reductase2 (anGR2) cDNA insert. Seeds from the two genotypes were compared morphologically. Glutathione levels in maturing and germinating seeds were measured by HPLC, and GR activity by native PAGE. Cytosolic glutathione was measured in situ by confocal laser scanning microscopy. Stress in the form of natural and accelerated ageing, and germination at high and low temperature and at low water potential was applied to both WT and anGR2 seeds to test vigor. Results show similar glutathione levels and GR activity (except during late imbibition) in WT and anGR2. In both genotypes, GSH/GSSG ratio increased and GR activity decreased during seed maturation. During imbibition, the glutathione pool becomes very reduced (<1% GSSG) and in WT seeds, GSH levels increase mostly by GSSG recycling. Cytosolic GSH in embryonic epidermal cells was estimated to be 1.1-1.6 mM. AnGR2 seeds aged faster, and were less tolerant of heat and drought stress than WT. Accumulation of glutathione during maturation indicated that glutathione is a major antioxidant in the seed during storage. Changes in GSH levels during imbibition coincided with ROS production during radicle protrusion. Under stress conditions, anGR2 seeds showed lower vigor, indicating perturbations in the ROS scavenging systems particularly GR2.
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    Improved regeneration and Agrobacterium-mediated transformation of wild strawberry (Fragaria vesca L.)
    Wadl, Phillip A. (Virginia Tech, 2005-12-14)
    The Rosaceae contains many important commercially grown fruit crops. No comprehensive genomics platform is currently under development for fruit crops, giving functional genomics studies with wild strawberry (Fragaria vesca L.) the potential of identifying genes important in fruit crops. Fragaria vesca has a small genome size compared to the cultivated strawberry, Fragaria à ananassa Duch. (164 vs. 600 Mbp per 1C nucleus). This feature, in addition to a short life cycle (12-16 weeks) and small plant size make F. vesca a good candidate for a model plant for genetic and molecular studies. The specific objective of this work was to develop an efficient high-throughput Agrobacterium-mediated transformation protocol to generate an insertional mutant population to support the justification of F. vesca as a model organism for rosaceous crops. The transformation techniques described by Alsheikh et al. (2002) and Oosumi et al. (2005) were modified and applied to a range of germplasm obtained from the USDA National Germplasm Repository. We found that the modifications made to the Alsheikh protocol were unsuccessful when applied to our germplasm. With the Oosumi et al. (2005) protocol, transformation efficiencies ranging from 11 to 100% were obtained for two accessions when explants were exposed to varying durations on TDZ containing medium during shoot regeneration. The transformation efficiency was given as the mean number of GFP+ plants obtained per primary explant cultured. Multiplex PCR, for amplification of the hptII and GFP genes, was performed on a random sample of GFP+ plants to verify insertion of the T-DNA. The statistical power of our experiment was insufficient to detect treatment effect but based on our findings the transformation efficiencies were high enough to justify PI 551572 for use in the high throughput transformations that are required to generate a population of insertional mutants large enough for gene discovery in F. vesca.
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    In silico cell biology and biochemistry: a systems biology approach
    Camacho, Diogo Mayo (Virginia Tech, 2007-06-01)
    In the post-"omic" era the analysis of high-throughput data is regarded as one of the major challenges faced by researchers. One focus of this data analysis is uncovering biological network topologies and dynamics. It is believed that this kind of research will allow the development of new mathematical models of biological systems as well as aid in the improvement of already existing ones. The work that is presented in this dissertation addresses the problem of the analysis of highly complex data sets with the aim of developing a methodology that will enable the reconstruction of a biological network from time series data through an iterative process. The first part of this dissertation relates to the analysis of existing methodologies that aim at inferring network structures from experimental data. This spans the use of statistical tools such as correlations analysis (presented in Chapter 2) to more complex mathematical frameworks (presented in Chapter 3). A novel methodology that focuses on the inference of biological networks from time series data by least squares fitting will then be introduced. Using a set of carefully designed inference rules one can gain important information about the system which can aid in the inference process. The application of the method to a data set from the response of the yeast Saccharomyces cerevisiae to cumene hydroperoxide is explored in Chapter 5. The results show that this method can be used to generate a coarse-level mathematical model of the biological system at hand. Possible developments of this method are discussed in Chapter 6.
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    Inheritance and expression of Cry3Aa and PVY-O coat protein transgenes in diploid and tetraploid potato
    Skoneczka, Jeffrey Allen (Virginia Tech, 2004-08-12)
    The potential benefits of plant genetic engineering for disease and pest resistance have been widely acknowledged in many studies, and although genetically modified crops are still encountering public wariness, these benefits warrant continued exploration. Because of its intrinsic economic benefits, the development of true potato seed (TPS) cropping systems has been instituted in many regions of the world. The incorporation of transgenic resistance could further the economic gain of farmers who are seeking ways to sustain their livelihood in the most efficient way possible. It is, however, largely unresearched how sexual hybridization of a transgenic crop would affect the behavior of a transgene in the resultant progeny. In the initial part of this study, transgenic lines were developed with a Cry3Aa transgene. These plants were then used in 4x-4x reciprocal crosses and 4x-2x hybridization schemes to determine the stability of the transgene after sexual hybridization. There was no observed parent of origin effect on transgene expression; however, a highly significant, non-mendelian inheritance of the Cry3Aa transgene was seen in the maternally inherited transgene of one set of progeny from a reciprocal cross. Additional transgenic lines of potato were developed with a PVY-O coat protein transgene. These plants were challenged with PVY-O and monitored for symptoms visually and for virus serologically. One transgenic line exhibited complete resistance to PVY-O while two others showed a delay in symptom occurrence. Further examination of the expression levels of the PVY-O coat protein transgene will be necessary to determine the type and usefulness of the observed resistance.
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    Metabolomics as a hypothesis-generating functional genomics tool for the annotation of Arabidopsis thaliana genes of "unknown function"
    Quanbeck, Stephanie M.; Brachova, Libuse; Campbell, Alexis A.; Guan, Xin; Perera, Ann; He, Kun; Rhee, Seung Y.; Bais, Preeti; Dickerson, Julie A.; Dixon, Philip; Wohlgemuth, Gert; Fiehn, Oliver; Barkan, Lenore; Lange, Iris; Lange, B. Markus; Lee, Insuk; Cortes, Diego; Salazar, Carolina; Shuman, Joel; Shulaev, Vladimir; Huhman, David V.; Sumner, Lloyd W.; Roth, Mary R.; Welti, Ruth; Ilarslan, Hilal; Wurtele, Eve S.; Nikolau, Basil J. (Frontiers, 2012)
    Metabolomics is the methodology that identifies and measures global pools of small molecules (of less than about 1,000 Da) of a biological sample, which are collectively called the metabolome. Metabolomics can therefore reveal the metabolic outcome of a genetic or environmental perturbation of a metabolic regulatory network, and thus provide insights into the structure and regulation of that network. Because of the chemical complexity of the metabolome and limitations associated with individual analytical platforms for determining the metabolome, it is currently difficult to capture the complete metabolome of an organism or tissue, which is in contrast to genomics and transcriptomics. This paper describes the analysis of Arabidopsis metabolomics data sets acquired by a consortium that includes five analytical laboratories, bioinformaticists, and biostatisticians, which aims to develop and validate metabolomics as a hypothesis-generating functional genomics tool. The consortium is determining the metabolomes of Arabidopsis T-DNA mutant stocks, grown in standardized controlled environment optimized to minimize environmental impacts on the metabolomes. Metabolomics data were generated with seven analytical platforms, and the combined data is being provided to the research community to formulate initial hypotheses about genes of unknown function (GUFs). A public database (www.PlantMetabolomics.org) has been developed to provide the scientific community with access to the data along with tools to allow for its interactive analysis. Exemplary datasets are discussed to validate the approach, which illustrate how initial hypotheses can be generated from the consortium-produced metabolomics data, integrated with prior knowledge to provide a testable hypothesis concerning the functionality of GUFs.
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    The Potential for Green Fluorescent Protein as a Screening Tool in the Production of Haploid Potato Plants
    Palumbo, Rose (Virginia Tech, 2003-12-17)
    A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. Hemizygous primary transformants (To) were produced from three leaf discs: 17 diploid plants from one leaf disc, three and nine tetraploids from the other two leaf discs. GFP expression was observed qualitatively under fluorescence microscopes and quantitatively with a GFP meter. Anther culture of tetraploids produced 29 plants, none with high levels of GFP. Segregation ratios for tetraploid T1 seedlings fit models for single duplex insertions (35 transgenic: 1 non) or double simplex insertions (15 transgenic: 1 non). Diploid T1 seedlings segregated for deleterious traits: dwarfed size and curled leaves, as well as the GFP transgene. Similar segregation patterns in diploid families implied that all diploids may have been from the same transformation event. The cumulative segregation showed the dwarfed and curled plants fit a single recessive gene ratio (3 normal: 1 mutant), and GFP fit a double-copy insertion ratio (15 transgenic: 1 non). There was substantial GFP silencing evidenced by the loss of expression in plants that had originally been selected for high GFP. However, six selections were found to be free of deleterious traits, consistently high expressers of GFP, and producers of stainable pollen with less 2n than IVP 101.
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    Proteome Profiling of Saccharomyces cerevisae stress response to Cumene Hydroperoxide (CHP)
    Tuli, Leepika (Virginia Tech, 2008-07-25)
    Oxidative stress, described as the state of disturbed intracellular redox balance, has been associated with several human conditions including ageing, apoptosis, cancer, autoimmune and neuro-degenerative diseases. Stress studies have shown that reactive oxygen species (ROS) and reactive nitrogen species (RNS) along with its intermediates can attack essential cell targets such as: DNA, proteins, lipids and carbohydrates, leaving behind dysfunctional biologic molecules. In effect, a cell's primary response is to involve several defense mechanisms that are under a complex and intricate regulatory control to repair any damages that may have occurred. Although several stress studies have been conducted in the past that have approached this biologically complex process step by step, application of a Systems Biology towards a comprehensive understanding is still emerging. The current objective of this project is to identify proteins that change in response to cumene hydroperxoide (CHP) treatment and in parallel make an attempt to uncover events and processes that are a part of CHP-induced oxidative stress response. From a systems biology viewpoint, the Yeast Oxidative Stress project will monitor response at three different levels: transcriptomics, proteomics and metabolomics, with dynamic changes being measured from 3 to 120 min after CHP addition. Data collected from the different levels will be integrated to accomplish a holistic viewpoint of stress response in the given system and to develop mathematical tools for modeling biochemical networks. Saccharomyces cerevisiae was chosen as a model, based on its availability of a completely mapped genome sequence with a collection of null mutants that was relevant to our fundamental research of stress response mechanism. Yeast, a simple unicellular eukaryote has been extensively used for applied studies and has proven to be indispensable for stress research. Information derived from this project can reveal response mechanisms used by higher eukaryotes, especially if via analogous signaling cascades that are comparable between organisms. Current research investigates an optimal workflow for generating 2D gel based protein expression data and identifying proteins that are induced by cumene hydroperoxide treatment. A non-targeted protein profiling followed by a 2-way ANOVA analysis provided a list of proteins that differ significantly between treatments. Protein identification provided relevant information on which proteins are affected by CHP induced stress response, including posttranslational modifications of peroxiredoxins. Redox active protein, Ahp1, was regulated post-translationally with sulfonic acid modification observed for its active Cys(62) residue.
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    Systems Biology in an Imperfect World: Modeling Biological Systems with Incomplete Information
    Pokrzywa, Revonda Maria (Virginia Tech, 2009-10-08)
    One of the primary goals of systems biology is to understand the complex underlying network of biochemical interactions which allow an organism to respond to environmental stimuli. Models of these biological interactions serve as a tool to both codify current understanding of these interactions as well as a starting point for scientific discovery. Due to the massive amount of information which is required for this modeling process, systems biology studies must often attempt to construct models which reflect the whole of the system while having access to only partial information. In some cases, the missing information will not have a confounding effect on the accuracy of the model. In other cases, there is the danger that this missing information will make the model useless. The focus of this thesis is to study the effect which missing information has on systems level studies within several different contexts. Specifically, we study two contexts : when the missing information takes the role of incomplete molecular interaction network knowledge and when it takes the role of unknown kinetic rate laws. These studies yield interesting results. We show that when metabolism is isolated from gene expression, the effects are not limited to those reactions under strong control by gene expression. Thus, incomplete understanding of molecular interaction networks may have unexpected effects on the resulting analysis. We also reveal that under the conditions of the current study, mass action was shown to be the superior substitute when the true rate equations for a biological system are unknown. In addition to studying the effect of missing information in the aforementioned contexts, we propose a method for limiting the parameter search space of biochemical systems. Even in ideal scenarios where both the molecular interaction network and the relevant kinetic rate equations are known, obtaining appropriate estimates for the unknown system parameters can be challenging. By employing a method which limits the parameter search space, we are able to acquire estimates for parameter values which are much closer to the true values than those which could be obtained otherwise.
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    Transposon Tagging in Strawberry and Potato and Characterization of Representative Strawberry Mutants
    Lu, Nan (Virginia Tech, 2013-09-25)
    Strawberry and potato are both important crop species in the world providing various nutritional values. The cultivated strawberry, Fragaria ananassa, is a fruit crop with a complex genome (2n=8x=56) whereas the diploid woodland strawberry, Fragaria vesca, has a smaller genome (2n=2x=14, 240 Mb) and lots of other qualities that make it a good model for genetic and genomic study, such as high yield of seeds and efficient transformation. Potato (Solanum tuberosum, 2n=4x=48) is an important vegetable crop in the world and is highly heterozygous. The successful sequencing of the homozygous doubled monoploid clone of potato provides good insight into the study of important genes in this species in improving the pest resistance and improving yield. One approach to characterize gene function in a model system is having large populations of T-DNA insertional or transposon tagged mutants. The idea of using AcDs construct to create transposon tagged mutant populations has also been applied in many species. Here we transformed two species, Fragaria vesca and a monoploid potato, Solanum phureja 1-3-516, which is the progenitor of the sequenced doubled monoploid clone, with the same AcDs construct, Ac-DsATag-Bar_gosGFP, to generate mutant collection, compare the marker gene performance and transposition efficiency, as well as characterizing phenotypic mutants with genes of interest. Transposants were found to reinsert to unlinked sites from the launch pad site in the strawberry genome, whereas in potato transposants tended to locate locally from the launch pad position when using the same construct. One transposon based activation tagging strawberry mutant, with its insertion in the promoter region of gene of interest in strawberry from the Ac-DsATag-Bar_gosGFP population was studied. In a segregating T2 population, expression level of the candidate gene, epidermis-specific secreted glycoprotein EP1 precursor, was 670 fold higher in petioles of homozygotes than in wild type plants, suggesting the function of this gene involved in maintaining mechanical strength of petioles. Since the often-used transposase gene was cloned from the monocot species maize, the efficiency of obtaining germinal transposants was many times lower than expected in order to saturate the genome for diploid species. In order to improve the chance of getting unique transposants, we attempted to codon optimize the transposase gene, as well as switching to microspore specific promoters that had been well characterized to control timing of expression of the transposase gene. Transposants were found in both T0 primary regenerates and anther culture derived potatoes using both the pAcDs-AtSCP and pAcDs-AmDEFH125 constructs. Sequencing of the empty donor site revealed that excision occurred in different cells during anther culture. A strawberry mutant with sugar transport deficiency due to T-DNA insertion near a sucrose transporter-2 gene showing stunted phenotype with increased level of anthocyanin was also characterized. The concentrations of sucrose, glucose, and fructose were significantly greater in source leaves of the mutant than wild type plants, suggesting these compounds might be substrates of this gene in transporting to sink leaves and roots.