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Development of a Transposon Based Activation Tagged Mutant Population in Tomato for Functional Genomic Analysis
Carter, Jared Daniel
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Tomato serves as an important model organism for Solanaceae in both molecular and agronomic research. With whole genome sequencing in progress, there is a need to study functional genetics through mutant lines that exceed the practical limitations imposed by the popular research cultivar, Micro-Tom. This project utilized Agrobacterium transformation and the transposon tagging construct, Ac-DsATag-Bar_gosGFP, to produce activation tagged and knockout mutants in the processing tomato variety, M82. The construct contained hygromycin resistance (hyg), green fluorescent protein (GFP), and maize transposase (TPase) on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 10,568 selfed and M82 backcross progeny was produced from the functional T0 line. This population was screened by spraying with 0.05% Liberty® herbicide, followed by a 100 mg/L hygromycin leaf painting procedure to select for Ds only (herbicide tolerant and hygromycin sensitive) individuals. The T-DNA genotype of Ds only plants was confirmed through multiplex PCR and the location of insertions within the genome was determined through TAIL-PCR. Resulting product sequences were blasted against the pre-publication tomato genome browser to determine insertion sites. A population of 309 independent transposants dispersed to all twelve chromosomes from the original insertion site on chromosome five has been developed. The transposon tagged lines are currently being immortalized in seed stocks.
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