Evaluation Of Fecal DNA And Its Availability In Przewalski's Horse (Equus Przewalskii)

Collection of tissue or blood samples often requires capturing the target animal,which inevitably has risks associated with injuries and difficulties of acquiring an adequate sample size. Non-invasive genetic sampling provides a new approach for research and management applications in wildlife conservation. However, for Przewalsi's horse(Equus Przewalskii) less non-invasive genetic sampling research was reported. DNA recovered from non-invasive samples such as feces has the typically drawbacks of low quantity and poor quality, which limited its application to the field research. This paper aimed to optimize the method of fecal DNA preservation and extraction and evaluated the availability of non-invasive genetic sampling in Przewalsi's horse.We evaluated and compared six combinations of three extraction and two preservation methods for fecal DNA, analyzed the effective storage time utilizing the 3 method, and constructed a technique system of fecal DNA preservation, extraction and PCR for Przewalsi's horse. In order to evaluate the availability of non-invasive genetic sampling, mitochondrial 12SrRNA was amplified and 4 microsatellite locus from domestic horse(Equus Caballus) were used to amplify 50 Przewalsi's horses.The main results are showed as follows:(1) Microsatellite amplification differed significantly between 3 storage types(P<0.05), with the ethanol the best. However, the interaction term was also significant(P<0.05), feflecting the fact that the optimal extraction method varied between storage types.(2) The highest amplification PCR success was obtained with samples stored in ethanol and extracted with the manual method(66.1%). Time dependent decay in amplification success was observed for samples stored in ethanol, drying, freezing, respectively at 9 months,6 months and 6 months.(3) CTAB,potato starch and BSA improved PCR by removing and inhibiting the co-purified PCR inhibitors in our manual stool DNA extraction method.(4) 4 microsatellite loci were used to conduct a general survey on the genetic of Przewalski's horse on 50 samples. A total of 16 distinct alleles were observed. The number of alleles per locus ranged from 3 to 5 with an average of 4. The mean expected heterozygosity (He) and observed heterozygosity (Ho) of these microsatellites were 0.703 and 0.575, respectively.