Zoology/Biology session

The African bullfrog (Pyxicephalus adspersus) has a ZZ/ZW sex chromosome system, however, a method for molecular sexing is yet to be developed. Males grow considerably larger than females, but phenotypic sex can only be determined following sexual maturation. However, with the aid of an affordable and easy-to-use genetic marker, the sex of even juveniles can be identified. In addition, molecular sexing could assist conservation biology research in the protection of African bullfrog populations. Additionally, it could also be used by breeders to identify the genetic sex of individuals at an early stage of ontogeny. In poikilothermic vertebrates, the sex-determining regions are less conserved than in warm-blooded animals, so different loci may often be responsible for sex-determination in these animals. Furthermore, in these taxa even closely related species may have different sex-determination systems. Consequently, markers developed for molecular sexing may only work within a single species or, in some cases, within a single genus. The aim of our research was to develop an inexpensive and easy-to-use PCR-based marker set that enables the molecular sexing of the African bullfrog. For the development of these genetic markers, we used the African bullfrog genome uploaded into the NCBI database, which was sequenced from a female bullfrog and thus contains both heteromorphic sex-chromosomes. The alignment of the sex-chromosomes, downloaded from the database, was performed using the Mauve tool in the software, Geneious Prime. We identified 168 potentially sex-linked sequences which served as the basis for the markers. From the 168 sequences, we designed primer pairs for 25 loci that contained insertion/deletion differences of appropriate size, which allows for the easy differentiation of PCR products on the agarose gel by size. Out of the 25 designed markers, we deemed 4 to be suitable for identifying genetic sex during testing. The PCR products were sequenced using the Sanger method to confirm their alignment with sequences uploaded into the database. We tested the markers on 26 individuals of known sex (14 male, 12 female) using DNA samples extracted from muscle tissue and blood. The muscle tissues were obtained from juvenile frogs, whose sex was determined by morphology and histology of the gonads. Blood samples were collected from mature individuals, whose phenotypic sex was determined based on morphological traits. For all four markers, the genetic sex determined using the PCR tests matched the previously established phenotypic sex, and no differences were observed in the sequenced amplicons aligned to the reference genome. Therefore, we can conclude that the developed markers show 100% sex linkage.