Fast Multiplex real time PCR method for sex-identification of medaka (Oryzias latipes) by non-invasive sampling

Abstract

Medaka fish (Oryzias latipes) has been widely used in fish screening and multi-generation tests to provide relevant data to assess impacts of endocrine disrupting chemicals (EDCs) in fish populations.The genotypic differentiation of Medaka sex allows diagnosing the sex reversal, and is required in current test guidelines (e.g. OECD TG 240, 2015). DNA isolation for genetic sex-identification requires sample collection, which has been normally conducted using invasive (fish sacrifice) or semi-invasive (fin-clip) procedures, which conflicts with the need for a fast, simple, and stress-free method. Swabbing skin mucus to collect DNA has been adopted in ecological studies of larger fish, however for smaller fish, it has to be established.To handle larger number of samples, real-time PCR represents a faster and sensitive method compared to conventional PCR. In this study, we aimed to develop a multiplex real-time PCR method for Medaka genetic sex-identification, using DNA sampled by swabbing as less invasive technique. In this approach, the male-determining gene DMY was used in combination with the cytochrome b housekeeping gene. • The method developed is a robust, rapid and a sensitive multiplex real-time PCR for Medaka genetic sex-identification. • This method allows the use of DNA isolated from fish by swabbing, as non-invasive sampling method. Method name: Multiplex real-time PCR, Keywords: Medaka, qPCR, Multiplexing, Non-invasive sampling