A Generalized Approach to Genetic Sex Detection of Uniparous Mammalian Species, With High Potential in Foetal Sex Determination Using Circulating Cell‐Free DNA of Maternal Plasma

Foetal sex determination of uniparous mammals with long gestation benefits the management of economically important domestic animals as well as captive wild populations, but the various methods currently available are limited to a few species. Based on the hypothesized presence of circulating cell-free foetal DNA (ccff-DNA) in the maternal circulation of relevant species, we aimed to develop a universal genetic assay that has the potential to determine foetal sex in a wide range of species. Highly conserved sequences on the Y chromosome and an autosome were identified and selected to serve as primer annealing sites for a male-specific marker and a reaction control marker, respectively. Primers were designed to amplify short target sequences to a maximum of 300 base pairs (bp) to ensure high sensitivity when applied to degraded foetal DNA. Ultimately, instead of a single universal assay, two sets of primers were designed separately for two groups of the relevant species, resulting in two generalized, highly sensitive duplex PCR assays, each consisting of an SRY (sex-determining region Y) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) marker. Following preliminary testing and PCR optimization, each assay provided nearly 100% specificity and sensitivity on genomic DNA samples of various species (20 species in total) and maternal plasma samples of an African elephant. Although further validation on maternal plasma samples is necessary, the developed assays have the potential to rapidly promote breeding-related activities of a wide range of species by providing a generalized and therefore inexpensive, rapid and convenient method for foetal sex determination.