Progress 09/01/08 to 08/31/11
Outputs
OUTPUTS: SNP polymorphism next to T-DNA insertions was used to place 108 unique pCAMBIA mutants of Fragaria vesca on the diploid strawberry map. Later flanking sequences from hi-TAIL PCR of new mutants were mapped by blast searching against the strawberry genome which had been assembled into pseudochromosomes based in part on SNP markers generated from the 108 original mutants. We identified homozygous lines of many mutants by segregation analysis of GFP expression or zygosity testing in a 3-primer PCR reaction. Mutants with extensive plasmid backbone integration defied TAIL-PCR identification of flanking sequence. We characterize tagged lines of F. vesca with abnormal phenotypes, including floral morphology (green petal), anthocyanin (white runner) and leaf morphology (curly leaf). Both the green petal and white runner phenotypes were not associated with their T-DNA insertions. Candidate gene analysis, verified with segregation analysis, was used to identify mutations causing green petal (37 bp deletion in an exon of a SEPALLATA3-like gene) and white runner phenotypes (20 bp deletion in the flavanone 3-hydroxylase gene). The T-DNA insertion in the curly leaf mutant was verified with complementation mutants. Pleiotropy, including abnormal pollen tube growth, seed set, petiolule length and epidermal morphology, was evident. The F. vesca genome and annotation resources were used heavily to characterize these mutants. For insertional mutagenesis in diploid strawberry by transposon tagging, we generated 180 unique primary transgenics of F. vesca Hawaii 4 transformed with pA-DsN-EG, harboring maize transposase and EGFP (Ac element) and nptII, p35S driving transposase and pmas driving EGFP (Ds element). Due to constitutive expression of transposase, 60 primary transgenics transposed somatically. Of remaining transgenics, 48 were potential launch pads with a single intact Ac/Ds T-DNA insertion. TAIL-PCR products placed launch pads on all seven strawberry linkage groups. T1 progeny analysis by multiplex PCR revealed 22-40% transposition in four putative launch pads on three linkage groups. Hi-TAIL PCR sequences from nested primers within Ds revealed transposition of Ds to distant sites in the strawberry genome. Some somatic transposition was found due to common integration sites among sibling transposants. We identified 68 independent transposants, most from a single line where the launch pad occurred in the 3'UTR region of a gametophycally expressed native gene. Three-primer (two flanking and one within Ds) PCR reactions could be used to identify homozygous T2 plants. We designed and constructed new transposon tagging constructs expected to restrict transposase expression to gametophytic tissue to reduce somatic transposition and the expense of identifying duplicate transposants. We hope to improve the expression of selectable markers. We entered the data on insertional mutants and transposants including seed availability into a Filemaker database. A forthcoming manuscript describing the transposon tagging saga in diploid strawberry will include a public database including mutant lines available, their insertion sites and whether seed is available. PARTICIPANTS: Richard Veilleux/Project director -- supervised graduate students and technicians; Vladimir Shulaev/co-PI -- co-supervised two graduate students, moved from Virginia Bioinformatics Institute to the Department of Biology a North Texas University, a new partner institution; Yinghui Dan/co-PI -- working at partner organization, The Institute for Advanced Learning and Research, Danville, VA -- co-supervised one graduate student, supervised a technician; Sarah Holt/graduate student -- worked on mutant characterization, troubleshooting protocols, genome research, transformations; Robert Lindsay/graduate student -- managed strawberry populations in tissue culture, growth chambers, greenhouse; Nan Lu/graduate student -- transformations, transposon tagging, TAIL-PCR Juan Jairo Ruiz-Rojas/graduate student -- sample collection, SNP analysis, mapping, TAIL-PCR, manuscript preparation; Kerri Mills/technician -- sample collection, TAIL PCR, multiplex PCR, greenhouse and growth chamber duties, database entry, sample organization; Cherish Davis/technician -- database development; Kendall Upham/technician -- database entry, general laboratory and greenhouse; many Virginia Tech undergraduates -- plant maintenance, seed extraction, DNA extraction; routine PCR TARGET AUDIENCES: Graduate students enrolled in Advanced Plant Breeding and Genetics were engaged in the project as we developed laboratory exercises involving DNA extraction, multiplex PCR, TAIL-PCR and mutant analysis based on the ongoing work. PROJECT MODIFICATIONS: We hoped to generate a much larger population of transposon tagged lines for this project. There were two major setbacks, one that GFP expression in our strawberry plants was not visible. GFP was supposed to give us positive selection for transformation and then negative selection for transposition. However, it was useless. We could demonstrate that the GFP gene was there using PCR but there was obvious GFP expression in only one of the transposon-tagged plants. The p-CAMBIA insertional mutants in the first set of studies had good GFP expression during callus growth, seedling growth, in roots and flowers. Because of our experience with these , we were not expecting an expression problem in plants carrying the AcDs construct. To accommodate the lack of expression, we resorted to multiplex PCR on a massive scale for every T1 seedling of the single insertion putative launch pads. This required thousands of DNA extractions and multiplex runs. We streamlined the DNA extraction protocol using a Genogrinder so that we could process more than 100 samples per day and optimized the multiplex protocol using Bioline Immomix to yield reliable results for the presence of full and empty donor sites as well as the presence of NPTII and a native strawberry gene. The second setback was the common finding of somatic transposition of the Ds element so that many of our primary transgenics had already transposed on first analysis. Since transposition removed the promoter from transposase, these somatic transposants were not going to mobilize the Ds element again; therfore, they became inactive and stable mutant lines similar to insertional mutants. In transformed plants where somatic transposition had not occurred, many were eliminated due to multiple T-DNA insertions and others due to a total lace of movement of Ds in test sets of 24 T1 seedlings. Of the four functional launch pads, two generated many T1 seedlings bearing the same site of transposition, again suggestion somatic transposition. The finding that the best launch pad was situated in a gametophytically expressed gene led us to redesign the entire construct to use gametophytic promoters instead of constitutive ones to drive transposase. The truncated transposase was obviously highly active in the strawberry system, so we retained that in our new construct. Also we found that one of our AcDs transgenic lines where the Ds element was shown to have inverted, such that the p35S promoter was driving EGFP instead of transposases, had incredibly bright GFP expression. So, our new construct was designed to have a double p35S promoter to drive EGFP and shows good GFP expression in transient assays. this is the only use of the p35S promoter in the construct to eliiminate the possibility of competitive expression of transgenes. Unfortunately, the transformation protocol for strawberry is too lengthy (16-20 weeks under optimal conditions) for us to have demonstrated the activity of the new construct in strawberry before the project terminated but we were able to use the tried and tested parts of the current project to build what we hope will be a better transposon tagging construct for strawberry and other dicots.
Impacts
The graduate students and PIs working on this project were actively involved in sequencing and publishing the strawberry genome assembly, a far reaching new genomic resource for Rosaceae. The dissertations and manuscript published from this work represent some of the first to employ this resource in Rosaceae. We have learned that insertional mutagenesis where each independent mutant derives from a unique T-DNA insertion is prohibitively expensive in time and resources to ever result in a population of strawberry mutants sufficient to have wide genetic and genomic coverage. Transposon tagging is essential for this goal. Our Ac/Ds construct, although imperfect, yielded data that are encouraging and provided a proof-of-concept for generating a large mutant strawberry population. Gametophytic transposition occurred globally throughout the genome in the progeny of some of our transgenic launch pads and the insertion of the Ds element was preferentially into genic regions. Reconstruction of the plasmid to overcome the limitations (somatic transposition, poor visualization of GFP) of the construct used in this study should provide a means to this end. Three of the graduate students who worked on this project have completed their degrees. The two Ph.D. students are now employed as postdocs in prestigious plant genetics labs. The M.S. student is pursuing a doctoral degree.
Publications
- Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher AL, Jaiswal P, Mockaitis K, Liston A, Mane SP, Burns P, Daviw TM, Slovin JPB, N., Hellens RP, Evans C, Harkins T, Kodira C, Desany B, Crasta OR, Jensen RV, Allan AC, Michael TP, Setubal JC, Celton JM, Rees JG, P. WK, Holt SH, Ruiz Rojas JJ, Chatterjee M, Liu B, Silva H, Meisel L, Adato A, Filichkin S, Troggio M, Viola R, Ashman TL, Wang H, Dharmawardhana P, Elser J, Raja R, Priest HD, Bryant Jr. DW, Fox SE, Givan SA, Wilhelm LJ, Naithani S, Christoffels A, Salama DY, Carter J, Lopez Girona E, Zdepski A, Wang W, Kerstetter RA, Schwab W, Korban SS, Davik J, Monfort A, Denoyes-Rothan B, Arus P, Mittler R, Flinn B, Aharoni A, Bennetzen JL, Salzberg SL, Dickerman AW, Velasco R, Borodovsky M, Veilleux RE, Folta KM (2010) The genome of woodland strawberry (Fragaria vesca). Nature Genetics 43: 109-116.
- Veilleux RE, Mills KP, Lindsay RC, Baxter AJ, Lu N, Davis CM, Ruiz-Rojas JJ, Holt SH, Ferguson TJ, Pantazis CJ, Shulaev V (2011) Transposon tagging in diploid strawberry with an AcDs system. Poster presentation at the Plant & Animal Genome XIX Conference, Jan. 15-19, 2011, San Diego
- Veilleux RE, Mills KP, Lindsay RC, Baxter AJ, Lu N, Davis CM, Ruiz-Rojas JJ, Upham KT, Holt SH, Ferguson TJ, Pantazis CJ, Dan Y, Dickerman A, Shulaev V. (2011) Transposon tagging in the diploid strawberry. Oral Presentation at the American Society for Horticultural Science Annual Conference, Sept. 25-28, 2011, Waikoloa, Hawaii
- Holt SH, Shulaev V, Veilleux RE, Dan Y. (2011) Molecular characterization of an anthocyanin-deficient mutant in the woodland strawberry, Fragaria vesca. Poster presentation at the Sixth International Workshop on Anthocyanins, Sept. 11-14, Concord, NC
- Holt SH, Dan Y, Shulaev V, RE Veilleux. (2011) Flower and fruit developmental defects caused by a spontaneous DNA deletion in a SEPALLATA3-like gene in Fragaria vesca. Oral presentation at the 2011 Society for In Vitro Biology Conference, June 4-8, 2011, Raleigh, NC
- Lindsay, Robert Clark, 2010. Seed germination, kanamycin sulfate selection, and the influence of nitrogen treatments on an insertional mutant population of Fragaria vesca. M.S. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA
- Ruiz-Rojas, Juan Jairo, 2010. Characterization of T-DNA integration sites within a population of insertional mutants of the diploid strawberry Fragaria vesca L. Ph.D. Dissertation, Virginia Polytechnic Institute and State University, Blacksburg, VA
- Holt, Sarah Hudson, 2011. Genetic studies of phenotypic variants in the woodland strawberry, (Fragaria vesca). Ph.D. Dissertation, Virginia Polytechnic Institute and State University, Blacksburg, VA
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: The project has engaged four graduate students, three Ph.D. and one M.S. level. Three of the Ph.D. students presented their work at the XVIII Plant & Animal Genome Conference in San Diego, in January, 2010. One of the Ph.D. students completed his dissertation and defended it in November, 2010. He published two chapters of his dissertation in refereed journals. Two technicians have also been working on the project, one to develop a database and another to process plant populations through the greenhouse and lab. Nine undergraduate students and two high school students have been employed in various aspects of the project. PARTICIPANTS: Richard E. Veilleux, PI; Vladimir Shulaev, co-PI; Yinghui Dan, co-PI, Institute for Advanced Learning and Research; Allan Dickerman, co-PI, Virginia Bioinformatics Institute; Nan Lu, graduate student; Sarah Holt, graduate student; Juan Ruiz, graduate student; Kerri Mills, lab technician; Cherish Davis, database technician; Megan Salaj, undergraduate hourly; Nathan Presnell, undergraduate hourly; Benjamin Morris, undergraduate hourly; William Jordan, undergraduate hourly; Connie Fu, undergraduate hourly; Eric Jochum, undergraduate hourly; Evan Ware, undergraduate hourly; Frankie Powell, undergraduate intern; Alyson Estes, high school student; Sylvie Nowak, high school student; Yinghui Dan and Sarah Holt are at the Institute for Advanced Learning and Research in Danville, Virginia and are primarily supported on USDA VAW-2009-04069 The project has also been heavily involved in the Strawberry Genome Sequencing Consortium, a multi-institutional international group that has worked to develop a draft sequence of the diploid strawberry genome. TARGET AUDIENCES: The strawberry project was developed into a series of laboratory exercises to complement a graduate course in Plant Breeding at Virginia Tech. Two undergraduates were enrolled in the laboratory portion of the course, one of whom has since enrolled in graduate studies in plant breeding. PROJECT MODIFICATIONS: The original AcDs construct that was used to transform F. vesca was found to be somewhat deficient as a transposon tagging vector. Although we have been able to develop several hundred transposon tagged strawberry lines, many have been found to occupy identical transposition sites, hence they were the result of somatic rather than gametic transposition. GFP screening was also ineffective in plants harboring this construct, necessitating PCR verification of potential transposants. While continuing to characterize the transposants that we obtained, we have adopted two other strategies. One has involved transformation with a completely different transposon tagging construct obtained from Andy Pereira, a collaborator at the Virginia Bioinformatics Institute on another NIFA project. We are evaluating the ability of launch pads that carry this construct to transpose. The other strategy is to try to fix what is wrong with the original AcDs construct. We know that the transposase is effective in both excising Ds and that the Ds reinserts apparently randomly into the genome. We also know that the kanamycin selection is effective at the callus stage, the seedling stage and the young plant stage. What needs repair is the GFP expression and the timing of Ds excision. We hope to correct both of these problems by replacing the promoters in the AcDs construct, using a different constitutive promoter on the Ds element to drive GFP expression in launch pad plants. Also, replacing the constitutive promoter in front of transposase with a promoter specific to microspore development to restrict transposition to male gametophytic tissues, hopefully pollen generative nuclei. Although the project will be finished before we can fully evaluate this new construct, we feel that it will pave the way to further research in transposon tagging in dicots.
Impacts
Fragaria vesca was transformed with a transposon tagging construct harboring maize transposase and EGFP (Ac element) as well as nptII, p35S driving transposase and pmas driving EGFP (Ds element). Of 122 primary transgenics, 38 were potential launch pads, 30 were multiple insertions or chimaeras, and 54 exhibited somatic transposition. T1 progeny of 39 potential launch pads were screened by multiplex PCR for transposition of Ds. Evidence of transposition occurred in 14 putative launch pads; however, transposition was too low in most for efficient recovery of transposants. Four launch pads that occurred on three different linkage groups detected by hi-TAIL PCR from nested primers in the tpase gene exhibited greater frequencies of putative transposition, ranging from 22% to 34%. After self-pollination of the T0 launch pads, putative transposants in the T1 were identified by lack of a full donor site and presence of an empty donor site (EDS) along with the Ds element or simply the Ds element. Sequencing of hi-TAIL PCR products derived from nested primers within the Ds end sequence (either p35s at the left border or right border inverted repeat) revealed transposition of the Ds element to distant sites in the strawberry genome. From these four launch pads, 1,252 T1 plants have been analyzed and 337 (27%) found to be putative transposants. The mutant collection is being cataloged in a Filemaker Pro database. We have demonstrated that a transposon tagging construct comprised of elements derived from maize, a monocot, can function to generate transposon tagged lines in the dicot, Fragaria vesca. We developed SNP markers adjacent to T-DNA insertions and then used the SNP markers to anchor scaffolds generated by 454 sequencing of the diploid strawberry genome into a genetic map. We could then blast TAIL-PCR products obtained from transposant plants against the assembled strawberry genome to determine the movement of Ds elements from their original launch pad position. Movement of the transposon tended to be global throughout the genome rather than local nearby the original T-DNA position.
Publications
- Oosumi, T., Ruiz-Rojas, J. J., Veilleux, R. E., Dickerman, A. and Shulaev, V. (2010) Implementing reverse genetics in Rosaceae: Analysis of T-DNA flanking sequences of insertional mutant lines in the diploid strawberry, Fragaria vesca L. Physiologia Plantarum 140, 1-9.
- Ruiz-Rojas J.J., Sargent D.J., Shulaev V., Dickerman A.W., Pattison J., Holt S.H., Ciordia A. and Veilleux R.E. (2010) SNP discovery and genetic mapping of T-DNA insertional mutants in Fragaria vesca L. Theoretical and Applied Genetics 121:449-463.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: To characterize T-DNA insertion sites of transgenic Fragaria vesca, we used hiTAIL-PCR (thermal asymmetric interlaced PCR) to amplify flanking regions surrounding either the left or right border of the T-DNA. Primers were designed from F. vesca flanking sequences to amplify products from both parents, F. vesca 815 x F. bucharica 601, of a diploid mapping population. Polymorphism occurred as: presence/absence of an amplification product for 16 primer pairs and different sized products for 12 primer pairs. For 46 mutants where polymorphism was not found for PCR product size, amplification products of both parents were sequenced to reveal SNP polymorphism. A CAPS/dCAPS (cleaved amplified polymorphic sequences; derived CAPS) strategy was then applied to identify a restriction endonuclease that could cut the allele of one parental line to provide the polymorphism needed to map the SNP position of 74 of the T-DNA mutants. We regenerated 230 primary transformants bearing an AcDs construct with transposase driven by a p35S promoter and EGFP driven by a pMAS promoter. The Ds element carried promoters for both transposase and EGFP in addition to nptII as selectable marker. Intact T-DNA would be expected to express all three genes on integration into the strawberry genome whereas transposants would only express nptII. The T0 generation was screened by multiplex PCR for presence of: 1) a full donor site (FDS) using primers that amplified a 600 bp product spanning a segment of the transposase gene on the Ac element and the p35S promoter on the Ds element, and 2) an empty donor site (EDS) using primers that amplified a 1,000 bp product between transposase and EGFP, a distance too great for PCR amplification under the selected conditions unless the Ds element had excised. Of 230 primary transgenics, 149 were potential launch pads (FDS and kanamycin resistant), 30 were either multiple insertions or chimaeras (FDS, EDS and kanamycin resistant), 54 were somatic transpositions (kanamycin resistant with or without the EDS) and three yielded anomalous bands that did not represent an expected product. T1 progeny of 24 potential launch pads were screened by multiplex PCR for transposition of Ds. Of 543 T1 progeny, 184 (34%) had transposed. Hi-TAIL PCR was developed using sets of nested primers in p35S towards the right border and the inverted repeat towards the left border of the construct. Sequencing hi-TAIL PCR products derived from primers designed using Ds end sequence revealed transposition of the Ds element to new insertion sites in the strawberry genome in 12 confirmed transposants derived from seven different launch pads. A second AcDs construct (G38) was used in transformation experiments to derive 22 independently transformed T0 plants. We streamlined our DNA extraction protocol by using a GenoGrinder to replace the tedious grinding of leaf tissue with liquid nitrogen in a mortar and pestle. The protocol yields high quality DNA suitable for multiplex PCR and hi-TAIL PCR both of which have been converted to 96-well format. We developed a Filemaker Pro database to store information about our mutant collection. PARTICIPANTS: Richard Veilleux, Project Director, Vladimir Shulaev, co-PI, co-advisor to graduate students Holt and Lu, Allan Dickerman, co-PI, bioinformatics, strawberry genome annotation, Yinghui Dan, co-PI, co-advisor to graduate student Holt, Jeremy Pattison, co-PI, co-advisor to graduate student Ruiz, Kerri Mills, technician, molecular characterization of mutant lines, Cherish Davis, technician, greenhouse organization and Database, Sarah Holt, graduate student, mutant characterization, Juan Ruiz-Rojas, graduate student, mapping insertion sites of mutants, Nan Lu, graduate student, transformation, Robert Lindsay, graduate student, screening mutants, Nathan Presnell, undergraduate student, greenhouse and lab work, Kendall Upham, undergraduate student, DNA extraction, PCR, Megan Salaj, undergraduate student, greenhouse and lab work, Tuan Phan, undergraduate student, intern, Benjamin Morris, undergraduate student, mutant screening, Sylvie Nowak, high school student, intern, Institute for Sustainable and Renewable Resources, partner organization, houses co-PI Dan and graduate student Holt, provided a population of transformed strawberry plants, Virginia Bioinformatics Institute, partner organization, houses co-PIs Shulaev and Dickerman TARGET AUDIENCES: The project was featured in the Summer, 2009 issue of the Virginia Tech Research Magazine and the Virginia Tech College of Agriculture Insight Newsletter. PROJECT MODIFICATIONS: Little or no GFP expression in transformed plants carrying the AcDs construct has changed our selection scheme. We are routinely using multiplex PCR in 96-well format to select T1 plant for various components of the system to be able to identify transposants. We are also attempting to build better dual selection vectors and evaluate existing ones developed for other plant transposon tagging systems.
Impacts
The research project has involved coordinated activity among four graduate students, three technicians and five undergraduate students. An emphasis in the project has been to streamline slow tedious protocols, so that greater populations of plants can be handled for molecular screening. Three major accomplishments were made in this regard: 1) the strawberry seed germination and selection procedure was changed from growing seedlings on agar solidified medium in disposable Petri plates to placing them in liquid selection medium in flasks in a lighted incubator shaker. This has improved both germination rate and provided better antibiotic selection. 2) The DNA extraction protocol was changed to eliminate the need and expense of liquid nitrogen, resulting in faster and more economical processing of samples. 3) Natural variation for seed germination was studied in F. vesca allowing the selection of lines with both a higher rate and more uniform germination. Better understanding of the pipeline among participants in the project has resulted in greater efficiency - plants are handled all throughout their life cycle, including seed germination, growth chamber establishment, leaf sample collection for DNA extraction, bench arrangement in the greenhouse, and molecular analysis, in a 96 well plate format, facilitating efficiency and reducing error. The project is related to the strawberry genome sequencing effort (under separate funding). Participants in the project have access to the draft strawberry genome and use its increasing sophistication in their research. This resource has allowed us to develop a zygosity assay to screen T1 strawberry seedlings for homozygosity of a transgene insertion using primers surrounding the T-DNA. Flanking sequence from TAIL-PCR from one end of the T-DNA can be blasted against the strawberry genome, the scaffold with the highest e-value selected and primers developed from sequence expected to occur on either side of the T-DNA. A three primer PCR reaction with the two flanking primers and one within the T-DNA amplify one product in a homozygous transgenic plant, a different product in a wild type plant and both products in a heterozygous transgenic plant. The system is being used routinely to select T1 homozygotes for advancing the mutant lines without needing to plant or screen T2. In addition, the Genome Browser annotation on the draft strawberry genome has allowed us to identify candidate genes that occur near specific T-DNA integrations. The crossover between this project and the sequencing project has provided graduate students a rich opportunity to develop cutting edge skills for their future careers.
Publications
- Shulaev V., Korban S.S., Sosinski B., Abbott A.G., Aldwinckle H.S., Folta K.M., Iezzoni A., Main D., Arus P., Dandekar A.M., Lewers K., Brown S.K., Davis T.M., Gardiner S.E., Potter D. and Veilleux R.E. (2008). Multiple models for Rosaceae genomics. Plant Physiology 147:985-1003.
- Pantazas, C., Flinn, B., Veilleux, R.E., Pattison, J., Nessler, C., Shulaev, V., Dan, Y. 2008. Strawberry transformation using kanamycin selection. In Vitro Cellular and Developmental Biology-Plant 44: S65-S65.
- Veilleux, R.E., Ferguson, T.J., Ruiz-Rojas, J.J., Pattison, J., Dan, Y., Holt, S.H., Pantazis, C., Mills, K.P., Davis, C.M., Flinn, B., Nessler, C.L., Shulaev, V. 2009. Strawberry insertional mutants derived from three different vectors. Plant & Animal Genomes XVII Conference, San Diego, California. January, 2009
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