| Summary: | The sex of an animal influences its economic traits, especially in species displaying sexual dimorphism. The Chinese soft-shelled turtle, <i>Pelodiscus sinensis</i>, is an economically important aquatic species that shows significant male sexual dimorphism, with a large body size, faster growth, a thick and wide calipash, and lower body fat. In this study, ten male and ten female turtles were subjected to restriction site-associated DNA sequencing (RAD-seq) using the Hi-Seq 4000 sequencing platform to isolate female-specific DNA fragments. We identified 5967 bp and 6532 bp fragments using genome walking. Three female-specific markers designed from these two fragments were confirmed to separate the sexes of <i>Pelodiscus sinensis</i> perfectly. One of the female-specific markers showed dosage association in female and male individuals. Individuals from different populations (n = 296) were used to validate that the female-specific markers could identify the genetic sex of <i>Pelodiscus sinensis</i> with 100% accuracy. The results of the present study demonstrated that RAD-seq was useful to develop sex-related markers in animals, and verified that the sex determination system of <i>Pelodiscus sinensis</i> belonged to the ZZ/ZW heterogametic system. Importantly, the developed markers could lead to a method for sex-controlled breeding in the Chinese soft-shelled turtle.
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