DOI: 10.5061/dryad.b2rbnzst9
title: Data from: Establishment of transgenic <em>Drosophila suzukii</em> lines that express <em>phiC31</em> integrase and carry the <em>sepia</em> gene as a marker for transformation

description: 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.
description: # Data from: Establishment of transgenic *Drosophila suzukii* lines that
 express *phiC31* integrase and carry the *sepia* gene as a marker for
 transformation Dataset DOI:
 [10.5061/dryad.b2rbnzst9](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 *se*1 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: *
 [https://doi.org/10.1111/imb.70001](https://doi.org/10.1111/imb.70001)
 Data was derived from the following sources: * Data in this dataset was
 derived from the above publication and supplementary information.