Declaration Dr. Thomas H. Pringle - Buffalo Field Campaign

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fidelity to breeding areas may be high, there should be genetic differences among bison breeding groups of the Yellowstone population, and between YNP and GTNP. METHODS Sample collection and storage We collected fecal samples from bison within the geographic range of each breeding group during the 2005 and 2006 breeding seasons. We determined the relative age class of individuals sampled through field observations of horn length and width, body size, and condition (especially for older animals). Social dominance ranking was recorded for individuals who displayed obvious behavioral clues such as displacement of other bison from foraging patches or wallowing pits, false charges, challenges for mates, and leading groups of other bison. Most samples (~ 5 grams) were collected within 10-15 minutes of defecation and placed into vials containing approximately 20 ml of 95% ethanol, and placed into coolers for up to 8 hours before they were frozen. Fecal samples were stored frozen at -20º C for up to 1 year prior to extraction. Extraction All fecal extractions were carried out in a designated non-invasive laboratory. Sterile filter tips, transfer pipettes, and microtubes were used. The QIAamp © Stool Mini Kit (QIAGEN) was used to extract genomic DNA from all fecal samples according to manufacturer’s protocol with modifications. Negative extraction and PCR controls were used to monitor for possible contamination. 34
PCR amplification Primers BISCR-16348F 5'-CTACAGTCTCACCGTCAACCC-3' and BISCR- 16990R 5'-GATGAGATGGCCCTGAAGAA-3' (Shapiro et al. 2004; Vogel et al. 2006) were used to amplify a 470 bp segment of the bison mtDNA control region. PCR was carried out in 25 μl volumes containing; 8.95 μl sterile HPLC H2O, 2.5 μl Invitrogen © 10X PCR buffer, 1 μl dNTP’s, 0.5 μl of each primer, 2.5 μl BSA (2ng/μl), 1.25 μl MgCl (50 mM), 0.3 μl Invitrogen © Platinum Taq Polymerase (5 units/μl), and 7.5 μl of template DNA. Amplification was performed in an MJ Research PTC-200 Peltier Thermal Cycler using the following touchdown protocol: 94º C for 5 min, followed by one cycle of 94º C for 30 s, 60º C for 30 s, and 72º C for 30 s. For the subsequent 10 cycles, all conditions remained the same except that the annealing temperature decreased by 0.5 degrees per cycle. This was followed by 25 cycles of 94º C for 30 s, 55º C for 30 s, and 72º C for 30 s. The profile concluded with a single extension of 72º C for 5 min. Sequencing PCR products were purified using QIAquick © purification columns according to manufacturers’ instructions with one exception; the final elution was carried out with 20 μl of Buffer EB instead of the recommended 30-50 μl to compensate for potential low quantity template DNA . The amount of purified post-PCR product was quantified by fluorometry to insure that 5-10 ng/μl of amplified DNA was present for sequencing on the ABI 3100xl sequencer. Sequences were visualized, assessed for quality, and edited using Chromas 2.31 (http://www.technelysium.com.au/chromas.html). MEGA 3.1 (Kumar et al. 2004) was used to align edited mtDNA sequences with known bison haplotypes. 35
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02/14/2011 10:36 5206827080 Summer
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02/14/2011 10:35 5205827080 " PICTU
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02/14/2011 10:35 5205827080 PICTURE
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Below, analysis of complete bison m
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• The mutation rate in mitochondr
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possibility of mitochondrial dysfun
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arginine R100 provides an essential
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inbreeding, small herd sizes and ge
Page 18 and 19: 42. XB Q, Jianlin H et al. Assessme
Page 20 and 21: Table 3. Bison non-sporadic mitocho
Page 22 and 23: Table 6. A. Phylogenetic conservati
Page 24 and 25: AY748671 ..........................
Page 26 and 27: DEVELOPMENT OF FECAL DNA SAMPLING M
Page 28 and 29: Table of Contents Abstract………
Page 30 and 31: List of Figures 1-1 Map of YNP show
Page 32 and 33: Dedication The work presented withi
Page 34 and 35: Preface Dale Lott grew up on the Na
Page 36 and 37: principles of the historic American
Page 38 and 39: within a possible third location (M
Page 40 and 41: samples. However, repeated genotypi
Page 42 and 43: population structure within YNP bre
Page 44 and 45: The highly differentiated populatio
Page 46 and 47: Figure 1-1. Map of YNP showing loca
Page 48 and 49: INTRODUCTION Wild bison are at risk
Page 50 and 51: sample was extracted twice using th
Page 52 and 53: Fragment analysis was carried out o
Page 54 and 55: visualized, assessed for quality, a
Page 56 and 57: aligned with less than two out of t
Page 58 and 59: ears (Ursus americanus), and brown
Page 60 and 61: Figures: Figure 2-1. Flow diagram s
Page 62 and 63: Table 2-2. Amplification success (A
Page 64 and 65: Appendix 2-3. Allelic dropout (AD)
Page 66 and 67: strong fine scale genetic different
Page 70 and 71: RFLP PCR amplification was carried
Page 72 and 73: etween the Lamar Valley and Hayden
Page 74 and 75: and Lamar Valley, in comparison to
Page 76 and 77: The differences in haplotype freque
Page 78 and 79: Figures: Figure 3-1. Approximate lo
Page 80 and 81: Literature Cited Allendorf, F.W., R
Page 82 and 83: Côté, S.D., F. Dallas, F. Marshal
Page 84 and 85: Luikart, G., S. Zundel, D. Rioux, C
Page 86 and 87: Tedford, S. Zimov, and A. Cooper. 2
Page 88 and 89: general approach using mtDNA marker
Page 90 and 91: 2 K.C. Douglas et al. / Mitochondri
Page 93 and 94: mitochondrial genome is 16318-16323
Page 95 and 96: Bison Herd (bHap13 and bHap16), the
Page 97 and 98: spread distribution of bison prior
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