A simple cross-species method for molecular sexing of softshell turtles (Trionychidae)

Abstract

Accurate sex identification is essential for aquaculture, breeding management, and conservation, particularly in species lacking external sexual dimorphism during early development. Softshell turtles (family Trionychidae) are of high economic importance: their meat is prized in markets, their shells are used in traditional medicine, and large-scale farming supports rural economies. Male Chinese softshell turtles grow faster than females; therefore, developing reliable sex identification methods for these species is critical. Softshell turtles possess evolutionarily stable ZZ/ZW sex chromosomes, which provide a strong genomic foundation for molecular sexing. In this study, we developed a PCR-based method for molecular sexing across 5 Trionychidae species, targeting intronic sequence variation in the Ran gene. A single band (~250 bp) is amplified in ZZ males, while two distinct bands (~250 and ~600 bp) are produced in ZW females, due to size differences in the second intron. This diagnostic pattern enables clear and reliable sex identification. Using this method with a conserved primer pair, we successfully sexed individuals of the Chinese softshell turtle (Pelodiscus sinensis), Spiny softshell turtle (Apalone spinifera), and Florida softshell turtle (Apalone ferox). The assay is rapid and cost-effective and offers a cross-species approach for molecular sexing across softshell turtle lineages and a practical tool for aquaculture, selective breeding, and conservation genetics.