Evaluating the Genetic Health in Bison on the Pine Ridge Reservation, SD Tada R Vargas Oglala Lakota College Mentor: Alessandra Higa NIFA-USDA award # 2011- 38424-309 14

Overview Background Target Population Research Question Methodology Results Conclusion http://www.outsideonline.com/1899041/oglala-sioux-tribe-and-park-service-partnering-first-tribal-national-park

Cultural Significance http://www.sd4history.com/Unit3/buffalolesson

History

Diagram 1: Founder’s Effect Restoration Process Today, North American bison populations exhibit a range of genetic purity depending on the history and management of individual herds. Determining the purity of a population is vital because, at some point, the level of introgression will impact the behavior, reproductive viability, and overall genetic species of an individual. Today, North American bison populations exhibit a range of genetic purity depending on the history and management of individual herds. Determining the purity of a population is vital because, at some point, the level of introgression will impact the behavior, reproductive viability, and overall genetic species Graph 1: Bottle Necking Diagram 1: Founder’s Effect

Research Question: Hypothesis: Null Hypothesis: How has cattle introgression affected the genetic diversity in Bison on the Pine Ridge Reservation? Hypothesis: The genetic variation of this population will not be in equilibrium from generation to generation due to disturbing factors. Null Hypothesis: The genetic variation of this population will remain constant from generation to generation.

Oglala Sioux Parks and Recreation Association

Methodology Hair Follicle DNA Extraction Mitochondrial based PCR assay Nuclear based PCR assay Electrophoresis Genotyping Genetic Analysis GenePop: Hardy Weineberg, Relatedness, and Inbreeding Coefficient

Figure 2: Extracting DNA DNA Extraction What happens to the cell. Cell breaks open, lipids, proteins, and RNA removed and DNA is purified. Protease, RNase, TE Buffer Ethanol and NaOH Figure 2: Extracting DNA Figure 3: Extracted DNA

Polymerase Chain Reaction PCR PCRReagents: dNTPs, BSA, Taq, MgCl2, and water Figure 4: Aliquoting Master Mix Figure 5: Transferring DNA Figure 6: Thermocycler Figure 7: Heating and Cooling Process

Figure 9: Gel Electrophoresis Gel electrophoresis is a method used to separate and analyze DNA fragments, based on their size and charge. They are separated by applying an electric field, which moves the negatively charged molecules thru the gel. Of course, smaller molecules will move faster than larger ones and this is how it is separated by size. After we view the gel in the photodyne to verify a successful reaction. What time of light is used in photodyne? Figure 9: Gel Electrophoresis Figure 10: Photodyne

Maternally Inherited Introgression Figure 11: Controls Figure 12: Gel results for Mitochondria analysis

Table 1 Nuclear Primers of PCR Multiplex Analysis Paternally Inherited Introgression Table 1 Nuclear Primers of PCR Multiplex Analysis Figure 13 and Figure 14: Gel photo for Nuclear Panel Primers

Figure 16 and 17: GeneAnalyzer (Closed and Open) Genotyping Prepping samples for genotyping is much like PCR in that we subject samples to reagents only in PCR we are amplifying a desired fragment. In genotyping, using the Geneanalyzer, we are able to discern what that fragment looks like. In this case are they homozygous bison, heterozygous bison, or homo or heterozygous cattle Figure 15: Aliquoting dilute PCR product for genotyping Figure 16 and 17: GeneAnalyzer (Closed and Open)

Introgression Genotypes Plot 1: No Introgression Confirming Markers Primer: RM 185 *Bison at 92 bp *Cattle between 90 - 108 Genotypes Plot 2: Positive Cattle Introgression Primer: BM 4307 *Bison between 185 - 187 *Cattle between 183 - 199

Variant Alleles Genotype Plots 5: Primer CSSM42 Bison: 167 – 171 Cattle: 173 - 217 Genotype Plots 6: Primer BM4307 Bison: 185 – 187 Cattle: 183 - 199 Genotype Plots 7: Primer CSSM42 Bison: 167 – 171 Cattle: 173 - 217

Genetic Diversity Genotype Plot 8: Confirming Marker Primer RM500 Bp size Bison: 123 Cattle 125 - 135

Genetic Diversity GenePlot: Confirming Marker INRA 119 Bp size Bison: 119 - 130 Cattle: 132 - 138

Genetic Diversity Genotype Plot 9: Introgression Primer BM4307 Bison: 185 – 187 Cattle: 183 - 199

Conclusion Genotyping is necessary to determine the presence of introgression. Exact levels of introgression cannot be determined without genotyping the entire genome. Possible new alleles but cannot be determined without genetic sequencing Provide detailed knowledge to develop strategies to best suit management goals Beneficial to interspecies introgression for wildlife population management and species conservation.

Acknowledgements

Questions?