Identification of sex-specific molecular markers and development of PCR-based sex detection techniques in tropical sea cucumber (Stichopus monotuberculatus)

Abstract

Sex-specific markers are essential for understanding sex determination mechanism and developing sex detection techniques in aquatic animals. The tropical sea cucumber Stichopus monotuberculatus is a fledgling aquaculture species in southern China. In this study, a total of 399 candidate male-specific and 179 candidate female-specific sequences were obtained after genome assembly, read mapping, and sequencing depth and coverage analyses of the sequenced genomes from three males and three females. Based on the k-mer distribution and sex-specific sequence length distribution, S. monotuberculatus is speculated to belong to a male heterogametic sex determination system. Next, 10 female-specific and 10 male-specific sequences were chosen for a first round verification to identify the true sex-specific markers, and only the male-specific markers could be repeated by PCR examination. The primer pairs for male-specific sequences were subjected to a second round verification, and five of them distinguished the genetic sex of S. monotuberculatus with accuracies of near 100%. Based on one of the male-specific markers (SM-M3), we constructed a rapid and nonharmful PCR-based method for sex identification that was suitable in different geographical populations, and it was successfully applied to distinguish female and male S. monotuberculatus before they were used for artificial spawning. Overall, this study provided evidence of the male heterogametic sex determination system and established an effective way to identify sex in sea cucumbers, and these results may contribute to the aquaculture of these economic species.