ZFX and ZFY gene sequences: use for molecular sexing European rabbits, European brown hares and mountain hares and perspectives for sex determination of other leporid species

We have developed a new molecular sexing method for three leporid species (Oryctolagus cuniculus, Lepus europaeus and Lepus timidus) based on the analysis by PCR-RFLP of point mutations that differentiate the ZFX and ZFY gene sequences. Polymorphic positions on the X and Y chromosomes of male samples were recognizable as double peaks in the sequencing chromatograms. Comparison of these sequences with those obtained from the females of O. cuniculus, L. europaeus and L. timidus allowed us to deduce the sequence of the Y fragment for these species. Analysis of the obtained sequences revealed an AluI restriction site in the female but not in the male sequences and a BglII restriction site in the male but not in the female sequences. A HinfI restriction site was present only in male O. cuniculus sequences but not in the female fragment. Digestion of the amplified fragments obtained in all collected samples for these three species produced the expected fragments for all analysed samples (70 European rabbits, 37 European brown hares and 24 mountain hares), for which sex was recorded during their collection proving a high accuracy of the methods. The method described here enables the identification or confirmation of gender of tissue samples from three species of Leporidae using restriction enzymes that cut male or female fragments. The specificity of this method means that the probability of incorrect sex identification due to polymorphisms within species that might involve the two gender specific restriction sites is low enough to be ignored. Then, we obtained sequence information for the ZFX/ZFY loci for other 6 leporid species (Bunolagus monticularis, Lepus americanus, Pentalagus furnessi, Romerolagus diazi, Sylvilagus floridanus and Sylvilagus nuttallii) including a few threatened taxa. Several polymorphic sites can be identified between sexes and among species. However, as few samples were available for these species further investigation should be carried out to develop and evaluate appropriate sexing protocols.