A novel indel-based molecular sex identification system for oplegnathus fasciatus: Insights from ndc80 gene polymorphisms and aquaculture applications

Abstract

Oplegnathus fasciatus is a commercially important marine fish species, valued both in wild fisheries and aquaculture. It possesses a multivalent sex-determination system (X₁X₁X₂X₂/X₁X₂Y) and exhibits marked sexual growth dimorphism, with males demonstrating significantly faster growth rates. Sex-specific molecular markers are instrumental in advancing selective breeding strategies. In this study, whole-genome screening of O. fasciatus revealed a male-specific structural variant within an intronic region of ndc80: two insertions of 3 bp and 538 bp (totaling 541 bp), which were consistently absent in females. To facilitate practical application, we developed a PCR-based assay using a single primer pair that amplifies a conserved region flanking the insertion site. This assay reproducibly generates distinct banding patterns: males yield two fragments (208 bp and 749 bp), consistent with the 541 bp male-specific insertion, whereas females yield only the 208 bp amplicon. This method enables efficient, high-throughput sex identification in O. fasciatus without the need for sequencing. Using standard agarose gel electrophoresis, the assay reliably distinguishes sexes through clearly divergent banding patterns. Beyond applications in selective breeding, these sex-specific markers provide critical molecular insights into the sex determination mechanisms and the genetic basis of sexual dimorphism in O. fasciatus.