Supported by the NSF Plant Genome Program, we are in the process of constructing a transgenic transposon-tagging genetic resource to meet the functional genomics needs of the maize community. The resource is designed to mobilize a modified Ds element from different starting sites in the genome.
More than 150 Agrobacterium-transformed lines carrying a single copy of a transpositionally active marked Ds* element have been generated and the location of 80 active platforms has been mapped to date (http://www.acdsinsertions.org/Status/_2). The development and use of this novel transgenic reverse genetics resource is outlined in the ensuing paragraphs.
Since both parents of the Hi-II hybrid used for transformation are c1 mutants, we based our transposon system on the c1-m2 allele, which carries a Ds element in the third exon. A c1 wx-m7(Ac); R line was derived to assay mobility of the engineered Ds* transposons in Hi-II (which is c1; r). An advantage of a c1-based selection system is that most inbreds are c1, so researchers can cross the c1-m wx-m7(Ac) stocks directly to theirs in order to generate Ds*-tagged mutations of their gene of interest.
A pTF102-derived binary vector based on c1-m2 was synthesized. In TAG21, Ds* element carries the marker P(22-kD-zein)/GFP, which conditions green fluorescent endosperms under blue light. Germinal transpositions of Ds* are easily identified in single-copy transgenotes as revertant purple kernels (C’) that produce fluoresce green (retain Ds/GFP).
Over 15,000 C’ green fluorescent revertant kernels have been isolated from several platforms. All these individuals carry trDs* elements. The site of insertion of over 3000 trDs* elements has been determined by high throughput sequencing of 3-D transposant pools using the InsertionMapper bioinformatic pipeline specifically developed for this project. Their exact location in the maize genome can be found in our project's website (acdsinsertions.org). Other sequence-indexed Ds* insertions will be added to our website as they become available. Stocks carrying these Ds* insertions are being regularly deposited in the Maize Stock Center.