Molecular sexing eutherian mammals

Mammals can be molecular sexed by polymerase chain reaction (PCR) amplification of Y chromosome fragments or coamplification of homologous fragments from both sex chromosomes, which are discriminated by size polymorphism or Y-specific restriction digestion. Although coamplification of X and Y fragments is more reliable, size polymorphism in homologous fragments is uncommon and Y-specific restriction site identification requires screening with a battery of enzymes or cloning. Here we describe a simple approach, using 'double peaks' in the chromatogram upon direct sequencing of PCR products from males, to identify Y-specific restriction sites, and demonstrate its utility by application to a range of taxa.