Plant & Animal Genomes XIII Conference

January 15-19, 2005
Town & Country Convention Center
San Diego, CA

Transposon Mutagenesis For Cotton Functional Genomics

Robert J. Wright¹ , Kelly Asbill¹ , Harriet Bergeron¹ , Jacy Lewis¹ , Thea A. Wilkins² , Norma L. Trolinder¹

¹  Dept of Plant & Soil Science, Texas Tech University, Box 42122 Lubbock, TX 79409-2122

²  Dept of Agronomy & Range Science, University of California Davis, 1 Shields Ave Davis, CA 95616-8515

To implement reverse genetic strategies in cotton, we have adopted a transposon mutagenesis strategy based on the Dslox system, which combines the advantages of transposon-tagging using maize Ac/Ds elements for producing insertion mutants, and Cre-lox site-specific recombination for inducing gross chromosomal rearrangements. The Cre recombinase catalyzes recombination between lox sites (34 bp palidromic sequences), resulting in chromosomal rearrangements (deletions, inversions, translocations) ranging from a few kilobase pairs to centi-morgan (cM) in length. The Dslox mutagenesis strategy offers an unprecedented opportunity to unveil gene functions important to agronomic traits. The T-DNA of the binary vector for introducing Dslox insertions contains a T-DNAlox site, and a Ds element embedded in a selectable marker and fused to a second lox site. In F1 progeny derived from a cross between a single-insertion T-DNAlox-Dslox line and a 35S::Ac transposase transgenic parent, excision of Dslox (transposed or tr-Dslox) from the inactivated marker restores the marker phenotype and indicates transposition has occurred. Single-copy tr-Dslox F2 segregants, when crossed to the 35S::Cre recombinase transgenic parent, undergo site-specific recombination between lox sites of tr-Dslox and T-DNAlox elements, resulting in deletions, inversion or translocations, depending on the relative orientation of the two lox sites. Approximately 200 Dslox transgenic cotton (G. hirsutum L. cv. Coker 312) lines have been produced by the NSF Cotton Genome Project. Ac-mediated transposition of Dslox has been demonstrated in transient assays, establishing that the Ac/Ds transposon-tagging system functions in cotton.