Identification of sex-specific polymorphic sequences in the goat amelogenin gene for embryo sexing

Amelogenin (AMEL) is a conservedgene located on the sex chromosomes of mammals. Itis involved in the formation of enamel, which is thehard, white material that forms the protective outerlayer of each tooth. In this study, we first cloned anddetermined the intron sequences of the goat AMELXand AMELY genes from female and male ear tissues.The polymorphic AMEL alleles were further analyzedby PCR-based RFLP and Southern blot hybridizationanalyses. Results showed that intron 5 nucleotide sequencesof the goat AMELY gene contains multipledeletions/insertions and shares only 48.5% identity tointron 5 of the goat AMELX gene. Based on the polymorphicAMEL intron sequences, a set of sex-specifictriplex primers was designed to PCR amplify a singlefragment of 264 bp from the X chromosome of femalegoats and 2 fragments of 264 and 206 bp from the Xand Y chromosomes, respectively, of male goats. Anincreased sensitivity for sex determination was reachedwith a single blastomere at the blastula stage isolatedfrom goat embryos. A total of 43 goat embryos wereused to estimate a 100% accuracy rate of this methodconfirmed by chromosomal karyotyping and live births.The embryo sexing technique has been successfully appliedin different strains of goats including Alpine, Saanen,Nubian, and Taiwan goats.