Differentiating among horse (Equus caballus), donkey (Equus asinus) and their hybrids with combined analysis of nuclear and mitochondrial gene polymorphism

A novel and brief method of differentiating among horse (Equus caballus) and donkey (Equus asinus) and their hybrids (mule, E. asinus × E. caballus and hinny, E. caballus × E. asinus) with combined analysis of nuclear and mitochondrial gene polymorphism (CANMGP) was reported in the present report. A nuclear gene, protamine P1 gene of donkey was sequenced and compared with the known horse sequence from GenBank while a published equid mitochondrial gene, cytochrome b gene of donkey was compared with that of horse. In each of the two genes, a fixed nucleotide substitution within an exon that could be recognized by Dpn II restriction enzyme was found between the two species. Two pairs of primers were designed for amplifying the fragments within the two genes containing the informative nucleotide positions in 65 horses and 41 donkeys and 38 hybrids and conditions of polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) analysis were optimized. Horse, donkey and mule and hinny had their own specific cleavage patterns after the PCR‐RFLP analysis was performed, which made it very easy to identify them from each other. As multiplex PCR can be conducted with the two pairs of primers and only one restriction enzyme is involved in PCR‐RFLP analysis, the method described in the present study is a convenient way to identify horse and donkey and their hybrids. The idea involved in the method of CANMGP can be also used to differentiate other animal species or breeds and their hybrids.