Transposon tagging in diploid strawberry

Abstract

SummaryFragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T 1 progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T 0 launch pads, putative transposants in the T 1 generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T 1 plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T 1 plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T 0 generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3¢ UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T 2 transposon-tagged plants. The mutant collection has been catalogued in an on-line database.

Fragaria: A genus with deep historical roots and ripe for evolutionary and ecological insights

“…Compared with most other cultivated perennials, the plants are compact, easily grown under controlled conditions, and can be propagated both sexually and asexually (Slovin and Michael, 2011). An efficient transformation system for F. vesca has been developed (Oosumi et al, 2006) and transposon‐tagged mutants are available for reverse genetics applications (Veilleux et al, 2012). The genome sequence of F. vesca has already facilitated significant progress in functional genomics of flowering (Mouhu et al, 2013; Hollender et al, 2014) and fruiting (Kang et al, 2013), and these advances will ultimately be applied to crop improvement efforts (Folta, 2013; Longhi et al, 2014).…”

Genomics tools available for unravelling mechanisms underlying agronomical traits in strawberry with more to come

“…On a larger scale, functional genomics can rely on the analysis of mutant collections, which have been developed or are currently being produced. After generating T-DNA or transposon-tagged lines (Oosumi et al, 2010;Veilleux et al, 2012), collections of EMS mutants appear to be very promising (Denoyes, Slovin pers comm).…”

Application of an inducible transposon with anther culture in generation of di-haploid homologous mutants

“…Transposable element (TE) tagging has become a powerful tool to create mutants for isolating new genes in animal and plant systems (Lin et al 2006; Horie et al 2011; Veilleux et al 2012). Several experimental approaches have been used to develop transgenic plants with genes randomly tagged by insertion elements (Greco et al 2001; Hirochika 2001; Hirochika et al 2004; Izawa et al 1997; Jeon and An 2001).…”