Computational protocol: Penetrance of symbiont mediated parthenogenesis is driven by reproductive rate in a parasitoid wasp

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Protocol publication

[…] Total DNA was extracted from wasps using a Chelex method () as implemented by . Gene sequences from the single-copy Trichogramma pretiosum gene wingless, and the Wolbachia 16S gene were identified from the genome assemblies (GenBank Accession Numbers: JARR00000000 and LKEQ01000000, ()). Trichogramma pretiosum wingless was identified through BLAST searches of the genome, using the Trichogramma evanescens homolog as a query (GenBank Accession Number: GQ368153.1). Specific primers () were designed to amplify variable regions of these two genes, using primer3 (). Primer specificity was checked computationally with Primer-BLAST (), and against extractions of the moth host eggs, E. kuehniella, which has an orthologous copy of wingless, and is infected with its own strain of Wolbachia. qPCR was performed in 20 μl reactions containing 1× ThermoPol™ buffer (New England Biolabs, Ipswich, MA, USA), 0.4 μM each primer, 200 nM each of dATP, dCTP, and dGTP, 400 nM dUTP, 1 mM MgCl2, 0.5 × EvaGreen® (Biotium, Fremont, CA, USA), 1 U Taq polymerase (New England Biolabs, Ipswich, MA, USA), and 2 μl of sample. Reactions were denatured at 95 °C for 3 min, followed by 35 cycles of 95 °C for 20 s, 58 °C for 20 s, and 72 °C for 20 s. All samples were run in triplicate alongside calibration standards and negative controls on a Rotor-Gene® Q (QIAGEN). Relative Wolbachia titers were determined with the ΔΔCt method () with normalization to wingless. When testing titers in offspring, we did not correct wingless quantification for ploidy levels between males and females as there is evidence that most of the somatic tissues in males are diploid (). […]

Pipeline specifications

Software tools Primer3, Primer-BLAST
Application qPCR