Fecal Matters Matters Noninvasive Fecal DNA Sampling Methods

Fecal Matters: Matters Non-invasive Fecal DNA Sampling Methods for Conservation Genetics Studies of Bison In Grand Teton and Yellowstone National Parks Introduction Michael O’Brien University of Montana, Project TRAIN Undergraduate Research Fellow Florence Gardipee Ph. D. Student, Fish and Wildlife Biology, University of Montana Gordon Luikart, Ph. D. Faculty Affiliate, Division of Biological Sciences, University of Montana The American Bison (Bison bison), sometimes called “buffalo”, is North America’s largest terrestrial mammal. Millions of bison once roamed across a vast majority of North America, and were integral part, culturally and economically, to the lives of a majority of North America’s native peoples. During the 19 th Century, bison were almost hunted into extinction, and by 1890 less than 1000 remained. The few remaining bison where forced onto parks and reserves, or were domesticized when privately-owned herds were established. Domestication of bison eventually led to the development of a fertile hybrid offspring when cross-breed with cattle, called beefalo or cattalo. Today, only about 300, 000 bison remain in North America, 90% of which reside in private herds. Recent genetic studies of privately-owned herds of bison reveal evidence of high levels of hybridization with domestic cattle; there as few as 12, 000 to 15, 000 pure bison in the world. Of these, an estimated 4000 bison in Yellowstone National Park are descendents of the last wild herd that continuously ranged in the Greater Yellowstone Area. Relatively little is known about population structure and gene flow among bison populations within the Greater Yellowstone Ecosystem. Prior sampling of bison for genetic studies has been opportunistic, and limited to animals who were captured or slaughtered as they exited the park. Using noninvasive sampling for DNA studies, would not only allow for the expansion of sample size to include animals inside the park, but would also allow us to look at both the population structure and gene flow of these animals as well. For the purpose of this study we chose to use feces as a source of DNA. However, according to Taberlet et al. (1999), DNA amplification from fecal samples (as well as other noninvasive sample types) can present several challenges to successful genotyping, and potentially offer false results. DNA extraction from feces is due to epithelial cells sloughed from the intestinal lining during defecation. Prior non-invasive studies have been conducted mainly on species whose dung is in pellet form. The epithelial cells are washed off the outside of the pellet using a lysis buffer. However, bison do not defecate pellets, rather their feces is more of a “patty” form. I predict that the exterior of the dung pile will yield higher concentration of intact DNA than the interior of the pile. Abstract Noninvasive sampling for amplification of mitochondrial and nuclear DNA can be impractical because it may be costly, time consuming, and unreliable. However, some prior studies suggest that this can be overcome by well developed protocols for collection, extraction, and amplification. We are developing protocols for use in American Bison (Bison bison) from Grand Teton and Yellowstone National Park. We are attempting to amplify both mitochondrial and nuclear DNA using the fecal matter from bison with low genotyping error rates. We will use the protocols to access gene flow and population dynamics of bison in these areas. Results Preliminary results suggest that we were able to amplify both mitochondrial and nuclear DNA from the both the exterior and interior of the fecal deposition. We have successfully optimized our PCR protocol for our mitochondrial makers, and are presently optimizing our microsatellite markers. Thus far, we have found a that a touchdown annealing temperature of 65ºC with a decrease of -1ºC /cycle for 10 cycles worked best the first 10 out 12 loci that we screened. Bison Blood samples 10/25/05 Positive Control q q Follow DNA extraction protocols for the Qiagen QIAamp DNA Stool Kit q Polymerase Chain Reaction (PCR) was carried out using Primers and a modified protocol developed by Halbert Derr were used to amplify DNA using a MJ Research Thermal Cycler. q Run PCR product on Argarose Gels to visualize. Current management of wild bison focuses of the issue of brucellosis in the Greater Yellowstone Area as opposed to the conservation of the last free roaming bison in North America. Literature Cited Gardipee, F. , G. Luikart, F. Allendorf. 2005. Proposal for pilot study: The feasibility of non-invasive fecal DNA analysis for genetic studies in the Yellowstone bison herd. Bison Mt. Dna Fecal Gel 2/9/06 Halbert, N. 2003. The Utilization of Genetic Markers to Resolve Modern Management Issues In Historic Bison Populations: Implications For Species Conservation. Acknowledgments Research Mentors: Florence Gardipee, Dr. Gordon Luikart, & Dr. Fred Allendorf Funding: Project TRAIN Undergraduate Research Fellowship, provided by a grant from the National Science Foundation. NSF EPSCo. R, University of Montana Division of Biological Sciences, University of Montana Obtain ~5 g of feces from the exterior and interior of the dung pile and place them in separate leak proof containers filled with 17 m. L of 95% Ethanol. Pipette ~2 m. L of the fecal/ethanol mixture into a 2 m. L eppendorf tube and centrifuge at full speed (14000 rpm) for around 3 minutes. Pipette of the ethanol. Due to the differences in seasonal forage quality, summer as opposed to winter, results are still inconclusive as to which part of the fecal pile yield the greatest quantity and quality mitochondrial and nuclear DNA. Fernando P. , T. N. C. Vidya, C. Rahapakse, A. Dangallo, and D. J. Melnik. 2003. Reliable Noninvasive Genotyping: Fantasy or Reality? Journal of Heredity 94(2): 115 -123 Collect fecal samples from Bison bison herds ranging in Grand Teton National Park, and Hayden and Lamar Valley in Yellowstone National Park. q Non-invasive Sampling may prove to be a feasible option for studying the genetics of Bison bison. Further investigation will show whether our protocols will be useful in conducting conservation genetic studies of these animals. Taberlet P. , L. P. Waits, and G. Luikart. 1999. Noninvasive genetic sampling: look before you leap. TREE 14: 323 -327. Materials and Methods q Discussion and Conclusions Patagonia Special Thanks to Rick Wallen, Jason Hicks, Erick Green, Scott Mills, and the Allendorf Lab and Staff Make your graphs big Enough so That you’ll really be able to see And read them.