Rapid Sex Identification in Spotted Knifejaw (Oplegnathus punctatus) Using tmem88 Gene Structural Variation Markers

Spotted knifejaw (Oplegnathus punctatus) is an economically important marine cultured species exhibiting a unique complex sex chromosome system (X₁X₁X₂X₂ in females and X₁X₂Y in males), with males possessing one fewer chromosome (2n = 47) than females (2n = 48) and an abnormally large Y chromosome. Additionally, males demonstrate significant growth advantages over females. Rapid and accurate sex identification is essential for effective culture management, selective breeding, and population control. In this study, we identified a homologous region of the tmem88 gene containing large DNA insertion markers on the X and Y chromosomes through whole-genome sequencing of O. punctatus. The X₁ chromosome harbors a 278 bp DNA fragment, whereas the Y chromosome contains a 1472 bp fragment, resulting in a 1194 bp size difference indicative of structural variation in the non-coding region of the tmem88 gene. We developed a rapid detection method based on this variation, utilizing a pair of primers that amplify two distinct bands (278 bp and 1472 bp) in male (X₁X₂Y) individuals and a single 278 bp band in female (X₁X₁X₂X₂) individuals when analyzed by agarose gel electrophoresis. This method enables efficient and accurate sex differentiation in O. punctatus, significantly reducing the time required for identification and enhancing detection efficiency. This study provides a valuable tool for the rapid identification of sex in O. punctatus, facilitating improved breeding strategies and supporting the large-scale production of high-quality fry.