A sequence-indexed collection of single Ds-GFP insertions spread throughout the maize genome
Dooner and Du (NSF award 1339238) generated sequence-indexed stocks of single transposed Ds elements tagged with GFP. The insertions are called “trdsg” for “transposed Ds-GFP". A mutant line in which a single gene has been disrupted gives biologists a powerful tool in understanding the function of that gene and are critical resources for elucidating gene function.
Using a combination of next-gen sequencing, 3-dimensional pooling, and a software package specifically developed for this project (Xiong et al., 2013), Dooner and Du generated a sequence-indexed collection of 14,184 Ds-GFP (Dsg) insertions scattered around the genome. For each insertion, 3’ flanking sequence, genome position and any tagged or nearby genes can be found in this table, or from Dooner and Du’s website: http://www.acdsinsertions.org/showt/Dsg. Insertions can be viewed on the B73 RefGen_v3 Genome Browser. Be sure to select track "Ac/Ds/GFP-Ds [from the Dooner Lab]", and stocks are available from the Maize Genetics Stock Center, searchable here. Enter "tdsg" in the simple search box.
Transposed Ds-GFP (trDsg) selection and use
The T-DNA launching platforms carry the c1-m(Dsg) engineered GFP tagged reporter allele (T0) and produce a green fluorescent, Ac-dependent spotted phenotype (T2) (see Figure below). Ac activity is provided by the native wx-m7(Ac) allele on chromosome 9 (T1). From any given T-DNA launching platform, Dooner and Du selected transpostions of Dsg (T3) as exceptional purple revertants that retain green fluorescence (C’+Dsg), so the spotted phenotype is lost at the outset. They crossed those selections to a c1 tester to confirm the concordancy of revertants (i.e., that the selection is heritable) and to determine the linkage between the transposed Dsg (trDsg) and the corresponding T-DNA launching platforms (T3). In the case of unlinked or loosely linked transpositions, they selected from the testcross progeny colorless (c), green fluorescent (Dsg) kernels (T4) for sequence-indexing and for seed propagation. If the trDsg was so closely linked that it produced no or very few colorless, green fluorescent seed in the testcross progeny, they selected purple (C’) green fluorescent (Dsg) kernels for sequence-indexing and for seed propagation. These are rare among materials sent to the Maize Genetics Stock Center. All sequence-indexed selfed progenies (T5) were deposited in the Maize Genetics Stock Center.
Please Note: If you order a transposed Ds-GFP (trDsg) insertion line from the Maize Genetics Stock Center note that the trDsg element is segregating in the progeny. Dooner and Du did not select for or against wx. If the waxy endosperm phenotype is still segregating, selecting for it should retain Ac, in case the researcher wants to initiate secondary movements of the mapped trDsg. Ac can transpose from wx-m7(Ac)leaving a defective wx allele, but such transpositions are rare. Selecting against wx should eliminate Ac, unless Ac transposed from wx during the derivation of the trDsg stock. Presence or absence of Ac can be efinitively ascertained by crossing to the native c1-m2 allele (available from the Maize Genetics Stock Center; search here; search for "tdsg").
InsertionMapper: a pipeline tool for the identification of targeted sequences from multidimensional high throughput sequencing data. Wenwei Xiong, Limei He, Yubin Li, Hugo K Dooner and Chunguang Du. BMC Genomics 2013, 14:679.
Gene tagging with engineered Ds elements in maize. Li, Y., G. Segal, Q. Wang, and H. K. Dooner. 2013 Methods in Molecular Biology: Plant Transposable Elements, 1057: 83-99. T. Peterson, ed. Springer Science & Business Media, NY. The final version of the book chapter is available on Springer Link
The complete Ac/Ds transposon family of maize. Chunguang Du, Andrew Hoffman, Limei He, Jason Caronna and Hugo K Dooner. BMC Genomics 2011, 12:588.
Use of the Transposon Ac as a Gene-Searching Engine in the Maize Genome. Matthew Cowperthwaite, Wonkeun Park, Zhennan Xu, Xianghe Yan, Steven C. Maurais and Hugo K. Dooner. The Plant Cell, Vol. 14, 713-726, March 2002, Copyright © 2002