Transposon tagging in diploid strawberry

• Published in: Plant biotechnology journal 2012-10, Vol.10 (8), p.985-994
• Main Authors: Veilleux, Richard E., Mills, Kerri P., Baxter, Aaron J., Upham, Kendall T., Ferguson, Tammy J., Holt, Sarah Hudson, Lu, Nan, Ruiz-Rojas, Juan J., Pantazis, Christopher J., Davis, Cherish M., Lindsay, Robert C., Powell, Frankie L., Dan, Yinghui, Dickerman, Allan W., Oosumi, Teruko, Shulaev, Vladimir
• Format: Article
• Language: English
• Subjects: AcDs; Biological and medical sciences; Biotechnology; Crops, Agricultural - genetics; Diploids; Diploidy; DNA Transposable Elements; Exons; forward genetics; Fragaria - genetics; Fragaria vesca; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Genomes; Genomics; Insertion; Introns; Inverted repeat; Launching pads; Multiplexing; Mutagenesis; mutant collection; Plants, Genetically Modified - genetics; Pollination; Polymerase chain reaction; Rosaceae; Sequence Tagged Sites; Strawberries; T-DNA; transformation; Transformation, Genetic; Transposase; Transposition; Transposons; Zea mays - genetics
• ISSN: 1467-7644; 1467-7652
• DOI: 10.1111/j.1467-7652.2012.00728.x

Summary Fragaria 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. T1 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 T0 launch pads, putative transposants in the T1 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 T1 plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T1 plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T0 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 T2 transposon‐tagged plants. The mutant collection has been catalogued in an on‐line database.