Development of gender-specific PCR test for Salmonids

Abstract

Most of the aquaculture industry relies on the female production stock because male rapidly leads to sexual development which causes poorer flesh quality and has increased chances to disease susceptibility. The sdY gene was recently discovered as a master sex-determining gene in Rainbow trout and the gene is considered to be conserved in most of the salmonid species. Four species of Salmonid (Salmo trutta, Salmo salar, Salvelinus alpinus and Salmo marmoratus) was selected for this study. Partial mRNA sequences of the sdY gene are available of Salmo trutta, Salmo salar and Salvelinus alpinus at NCBI. These partial mRNA sequences were used to design primers with the aim to amplify and determine the full-length sdY mRNA (cDNA) of Salmo trutta, Salmo salar and Salvelinus alpinus. hiTAIL PCR and 3' RACE was performed to obtain 5' and 3' end of the sdY gene and based on this new sequence information, primers were designed for PCR-based and HRM analysis genotyping methods to differentiate male and female samples. Duplex and triplex PCR-based genotyping were developed. Both the duplex and triplex PCR-based genotyping methods do not able to differentiate the sex of the selected species. HRM analysis using the same sets of primers as in duplex and triplex PCR-based genotyping can readily able to differentiate the sex of the selected species. The developed methods of genotyping cannot be concluded as the best method until we will not test these methods with more sex identified samples of these species.