Detection of genetic sex using a qPCR-based method in the bigfin reef squid ( Sepioteuthis lessoniana)

Abstract

The bigfin reef squid (Sepioteuthis lessoniana) is a valuable commercial species, especially in East Asia. While small-scale cultivation exists, efforts to achieve large-scale aquaculture are underway. However, full-life cycle aquaculture remains challenging due to reproductive issues, particularly in later generations, where abnormal offspring production is common. One possible issue is sex reversal under culture conditions, possibly due to high temperatures and smaller egg sizes in later generations. Therefore, a reliable method to determine the genetic sex of bigfin reef squid is crucial. Analysis of the female and male genome coverage along the assembly confirms a ZZ/Z0 female hemizygous sex-determination system in bigfin reef squid. We identified Z chromosome linked regions and developed a quantitative PCR (qPCR)-based method to measure the copy number variance of these Zspecific regions, normalized by autosomal regions, with ratios of 1:0.5 in ZZ males and Z0 females, respectively. Among the primer pairs designed for qPCR, two primer pairs in Z-specific regions and one primer pair in autosomal region exhibited a single melting curve (indicating the predicted same fragment) across all lineages. This genetic sexing method was validated in samples from different populations across Taiwan, demonstrating its effectiveness in determining the genetic sex of bigfin reef squid. Our findings provide a valuable tool for aquaculture management and offer new insights into sex determination mechanisms in cephalopods.