Abstract
Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza sativa), mPing, and the genes required for its mobilization, were transferred to soybean to determine if it will be an improvement over the other available transposon-tagging tools. Stable transformation events in soybean were tested for mPing transposition. Analysis of mPing excision at early and late embryo developmental stages revealed increased excision during late development in most transgenic lines, suggesting that transposition is developmentally regulated. Transgenic lines that produced heritable mPing insertions were identified, with the plants from the highest activity line producing at least one new insertion per generation. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene. Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource.
Highlights
Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality
A transposon that exhibits many of the desired traits for transposon tagging is the mPing element from rice (Oryza sativa; Jiang et al, 2003; Kikuchi et al, 2003; Nakazaki et al, 2003). mPing is a 430-bp miniature inverted repeat transposable element that transposes at a high frequency and has reached high copy number in some rice cultivars (Naito et al, 2006)
In Arabidopsis, mPing was mobilized by expressing the ORF1 and TPase proteins from either Ping or the closely related Pong element (Yang et al, 2007)
Summary
Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource. Unlike most DNA transposons that often cause indels (called footprints) at the site of excision, mPing excision sites are repaired precisely at a high frequency (99% and 82%) in both yeast (Saccharomyces cerevisiae; Hancock et al, 2010) and Arabidopsis (Yang et al, 2007), respectively These transposition characteristics indicated that mPing could be suitable for transposon tagging, with the caveat that heritable insertions had not been shown in hosts other than rice
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