A minimally-invasive protocol for molecular sex identification of the Chinese soft-shelled turtle Pelodiscus sinensis

Chinese soft-shelled turtle (Pelodiscus sinensis) adults exhibit sexual dimorphism, but sex identification during early life stages (hatchlings and juveniles) is difficult because of the absence of sexually-dimorphic morphological characteristics and the unavailability of a suitable non-invasive and simple alternative method. We hypothesized that differing copy numbers of the 18S rRNA gene on heteromorphic Z/W sex chromosomes would be a suitable sex marker. First, we had to establish a non-invasive method for DNA extraction. We knew from practical experience that taking a small tissue clip off the edge of the calipash creates a small wound that heals very quickly, but the suitability of this tissue for DNA extraction had not previously been confirmed. Thus, we compared 3 methods of genomic DNA extraction from a small calipash clip: phenol-chloroform, ammonium acetate, and a commercial kit. All 3 methods produced satisfactory DNA yields, both quantitatively and qualitatively. We sequenced the entire 18S rRNA gene and found that it is highly conserved, both intraspecifically and interspecifically. We then used quantitative polymerase chain reactions to quantify the copy number variation of the 18S gene between males (ZZ) and females (ZW). Females and males exhibited non-overlapping distributions, and females had similar to 10x more gene copies than males. We compared these data with the results of sex identification based on dimorphic morphological characteristics (63 adults) and histological examination of gonads (19 juveniles). Agreement between the methods was 100%. Therefore, here we present a minimally-invasive, simple, fast, cheap, and accurate method for DNA extraction and molecular sex identification of P. sinensis. This method may also be applicable to other related species that have sexually-dimorphic chromosomes.