Development Of A Species Specific Aflp-based Scar Probe For Black Muntjac (muntiacus Crinifrons)

Black Muntjac (Muntiacus crinifrons), a rare and endangered deer endemic to China, holds a high value in academic research. The species is currently regarded as the state key protected wild animal due to its limited distribution range and small population in the wild. However, because of the economic value of the meat, antlers and skin, the species has been hunted as the main targeted animal in traditional hunting practices and is still suffered from poaching at the present time. Usually, in order to escape from punishment, the poached black muntjac was peeled and sold together with carcasses of other animals, such as Chinese muntjac or livestocks. To provide a convenient, reliable and effective tool for authentication of tissue specimen such as, meat, skin and other tissues of the species, in this study, a Sequence Characterized Amplified Region (SCAR) derived from the black muntjac species-specific Amplification Fragment Length Polymorphism (AFLP) marker has been developed.Initially, a 707-bp species specific DNA fragment of black muntjac was detected by an AFLP primer pair (Ep7/Mp8). Then, the DNA fragment was purified, cloned and sequenced. Subsequently, based on the specific AFLP fragment sequence, a black muntjac-specific primer pair (P-F/P1-R) was designed by using Primer Premier 5.0 and Oligo 6.0, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified across all the samples, PCR amplification with the cyt b universal primers as positive control. As expected, out of the all forty-three DNA samples representing nine different species, all the twenty-five black muntjac samples presented two bands (about 298bp for SCAR and 466bp for cyt b). But the others failed to produce the SCAR with merely showing one band, the 466-bp cyt b fragment. The results indicated that the black muntjac-specific AFLP marker has been converted into a SCAR marker successfully and the SCAR primers were highly species-specific. Just based on the presence and absence of the SCAR band, we could discriminate black muntjac from other species rapidly and accurately. Therefore, the SCAR primers developed in this study will provide an effective tool for forensic authentication of tissues from black muntjac and strengthen the detection of poaching. And also, the SCAR probe may be useful for population survey in the wild through noninvasive sampling method.