Data from: Establishment of transgenic Drosophila suzukii lines that express phiC31 integrase and carry the sepia gene as a marker for transformation
Data files
Sep 02, 2025 version files 72.04 KB
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README.md
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Table_S1_revised-Final.xlsx
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Table_S2_revised-Final.xlsx
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Table_S3_revised-Final.docx
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Table_S4_revised-Final.docx
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Abstract
Many eye colour mutants have been identified in Drosophila melanogaster. Mutations in the sepia gene result in brown eyes due to a lack of PDA synthase, which is essential for production of the red drosopterin eye pigment. We previously used CRISPR/Cas9 to target the PDA synthase gene to establish sepia mutant strains for Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), an invasive global pest of soft skinned fruits. The fecundity and fertility of some of the sepia mutant strains were similar to wild‐type. The goal of this study was to determine if the sepia gene could be used as a marker to identify transgenic D. suzukii. By using the sepia gene as a marker, we successfully developed lines expressing Streptomyces phage phiC31 integrase in the germline. For most of these lines, hemizygotes exhibited complete rescue of the sepia eye colour and relatively high levels of phiC31 RNA in ovaries. In contrast, lines with partial rescue showed low levels of sepia RNA in heads and phiC31 RNA in ovaries. These findings suggest that the sepia gene is an effective marker for D. suzukii transgenesis, and its relatively small size (1.8 kb) makes it advantageous when assembling large gene constructs. The phiC31 integrase lines established in this study should serve as a valuable resource for future genetic research in D. suzukii, including the further development of strains for genetic biocontrol.
Dataset DOI: 10.5061/dryad.b2rbnzst9
Description of the data and file structure
To measure the expression levels of phiC31 and sepia, primer pairs PhiC31qPCR1F/PhiC31qPCR1R, Seg2RTF/Seg2RTR, and ActinRTF3/ActinRTR3 were designed, as specified in the supplementary materials (Table S4). The sepia primers were designed to amplify and detect the active sepia mRNA by using a forward primer that ends in sequence deleted in se1 and by taking advantage of the lack of 3' to 5' exonuclease activity in Taq polymerase (Figure S4). Quantitative PCR was conducted using the Luna® Universal qPCR Master Mix (Cat# M3003L, New England Biolabs) and cDNA synthesized in a CFX384 Touch™ real-time PCR detection system (Bio-Rad, Hercules, CA, USA). All reactions were performed in triplicate for both biological and technical replicates. The 2−ΔΔCt method (Livak and Schmittgen 2001) was used for data analysis, with actin used as a reference gene (Zhai et al. 2014) to normalize sepia and phiC31 RNA levels.
Supplementary tables S1 and S2 show the raw data for the real time PCR experiments that were carried out to quantify sepia and phiC31 RNA levels. If n/a is listed the data is not available as the technical replicate failed. The mean was calculated from the other replicates. Supplementary Tables S3 and S4 list the oligonucleotide primers used in this study. The Supplementary Figures show images of flies and DNA sequences that support the genome locations of the transgenes and edits in the targeted gene.
Files and variables
File: Figure_S1.tif
Description: FIGURE S1. Transgenic flies showing full and partial rescue of sepia eye color phenotype. Male (A) and females (B) from the NPS lines that have the SE+ DNA construct. Male (C) and females (D) from the INPS lines that have the SE+attP DNA construct. NPS4A48, INPS2 and INPS3 shows a partial eye color rescue phenotype compared to the other fly lines. The mutant flies with the sepia eye color are from the se1 line and WT is wild type.
File: Table_S1_revised-Final.xlsx
Description: Real time PCR Ct values for sepia and actin plus relative expression calculations
Variables
- Sample Name: RNA extraction sample name
- Target: Target gene name
- Ct1, Ct2, Ct3: Technical replicates for target gene
- Average Ct: Mean Ct of target replicates
- Reference: Reference gene name
- Ct1, Ct2, Ct3: Technical replicates for reference gene
- Average Ct: Mean Ct of reference replicates
- deltaCt: ΔCt (target gene Ct – reference Ct)
- delta delta Ct: ΔΔCt (sample ΔCt – mean ΔCt of reference)
- Fold Change (2−ΔΔCt): Relative expression fold change
File: Table_S3_revised-Final.docx
Description: Oligonucleotide primers were used in this study.
Variables
- Primer Name: Oligonucleotide primer name
- Sequence (5′→3′): DNA sequence
File: Table_S2_revised-Final.xlsx
Description: Real time PCR Ct values for phiC31 and actin plus relative expression calculations
Variables
- Sample Name: RNA extraction sample name
- Target: Target gene name
- Ct1, Ct2, Ct3: Technical replicates for target gene
- Average Ct: Mean Ct of target replicates
- Reference: Reference gene name
- Ct1, Ct2, Ct3: Technical replicates for reference gene
- Average Ct: Mean Ct of reference replicates
- deltaCt: ΔCt (target gene Ct – reference Ct)
- delta delta Ct: ΔΔCt (sample ΔCt – mean ΔCt of reference)
- Fold Change (2−ΔΔCt): Relative expression fold change
File: Figure_S4_copy.pdf
Description: Seg2RTF specific primer designed to amplify the wild type sepia gene and not the se1 mutant. Alignment of wild type (WT) and se1 mutant DNA sequences with location of the Seg2RTF primer shown.
File: Figure_S2_copy.pdf
Description: Sanger sequencing results of DNA fragments obtained by splinkerette PCR. SE+ lines (A) SE+attP lines (B). 5’PBAC or 3’PBAC sequences were identified in the sequences and are indicated.
File: Table_S4_revised-Final.docx
Description: Oligonucleotide primers used for qPCR
Variables
- Primer Name: Oligonucleotide primer name
- Sequence (5′→3′): DNA sequence
- Primer efficiency: Real-time PCR efficiency
- R² Value: Coefficient of determination
File: Figure_S3_copy.tif
Description: The INPS4 Y-linked line shows partial rescue of the sepia eye color in males. (A) Eyes of the INPS4 line males and females. (B) Sanger sequencing results of DNA fragments obtained by splinkerette PCR. 5’PBAC sequence was identified in the sequences and is indicated.
Code/software
The figures can be viewed using a pdf reader. Tables S1 and S2 can be opened in Excel and Tables S3 and S4 using Word.
Access information
Other publicly accessible locations of the data:
Data was derived from the following sources:
- Data in this dataset was derived from the above publication and supplementary information.