Fig 1.
Constructs used for generating transposon-tagged lines in tomato.
A. Ac and Ds constructs used for insertional mutagenesis in rice. B. Modified Ac and Ds constructs adapted for insertional mutagenesis in tomato. Abbreviations: p35S: CaMV 35S promoter; Ac-TPase: Ac transposase; T-NOS: Nopaline synthase terminator; pUbi: Maize ubiquitin 1 promoter; GFP: Green fluorescent protein gene; YFP: Yellow fluorescent protein gene; 4X EN: 4X 3CaMV5S enhancers; Ds3′: 3′ end of the Ds element; Ds5′: 5′ end of the Ds element; At-Ubi: Arabidopsis thaliana ubiquitin 3 promoter.
Fig 2.
Imaging of GFP and RFP fluorescence in Ac- and Ds- plants.
Wild-type (WT) plants (cv. Arka Vikas) and transgenic GFP-expressing plants in the Moneymaker background were used as negative and positive controls, respectively. The emission of GFP and RFP fluorescence confirms the presence and expression of the respective transgenes. A-B. Confocal laser scanning microscopy images showing GFP (A) and DsRed/RFP (B) expression in T0 Ac and Ds lines. C. Top panel: images of leaves taken with a GFP filter; bottom panel: corresponding bright-field images. The blue fluorescence indicates GFP signal saturation. Ac-24A T2 is a Southern-positive line. D. Top panel: images of WT (left) and MM-GFP (right) seedlings under an RFP filter. Bottom panel: RFP fluorescence in Southern-positive Ds-10-2 T1 seedlings. E. Top panel: images of WT (left) and MM-GFP (right) seedlings under a GFP filter. Bottom panel: GFP fluorescence in seedlings of Ac T3 lines. Images in panels C–E were captured using the Kodak imaging station.
Fig 3.
In vivo imaging of RFP and GFP fluorescence in F1 and F2 seedlings.
A. Fluorescence imaging of F1 seedlings from the crosses Ds-2 × Ac-24A3, Ds-2 × Ac-9G9, and Ds-3 × Ac-2C4, captured under a GFP filter (left panel) and an RFP filter (right panel). Seedlings were illuminated using a UV lamp. B. Fluorescence Imaging of F2 seedlings from the crosses Ds-2 × Ac-24A4, Ds-3 × Ac-2C4, and Ds-2 × Ac-9E1, using the same setup with GFP (left panel) and RFP (right panel) filters.
Fig 4.
Analysis of Ac-Ds segregation in the F2 generation.
A. PCR amplification using GFP-specific primers. A 331 bp amplicon indicates the presence of either both Ac and Ds elements or Ac alone. A white asterisk marks the absence of the GFP amplicon. B. PCR amplification using RFP-specific primers. A 500 bp amplicon signifies the presence of the mobilized Ds element only. A red asterisk*) denotes samples where only the RFP amplicon was detected.
Fig 5.
Inverse PCR Analysis of Ds-tagged DNA in GFP ⁻ /RFP ⁺ Ds F2 lines.
A. Inverse PCR products generated from DNA isolated from leaves of Ds-only plants. B. Gel-purified amplicons of flanking sequences obtained post-PCR. Note: Asterisks indicate the amplicons of flanking sequences in different samples. M: 1 Kb Marker. Lanes 1-16, Ds-only plants.
Fig 6.
Schematic representation of Ds transposon distribution across the twelve tomato chromosomes.
Transposon insertions were detected on 11 of the 12 chromosomes. Blue asterisks indicate insertions within genic regions, while black asterisks represent intergenic insertions. Purple asterisks denote insertions with unknown launch sites. Red asterisks on the left side of chromosomes 1 (Ds-2), 3 (Ds-10-2), and 9 (Ds-8) indicate the positions of known launch sites. The schematic incorporates data from both sets of crosses. Detailed genomic coordinates of the insertion sites are listed in S1 Dataset.