Declaration Dr. Thomas H. Pringle - Buffalo Field Campaign

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02/14/2011 10:35 5205827080 " PICTURE ROCKS HDWR nine standard methods of biomedical genetics, an academic field under development since 1949. 9. One of the pUblications I relied upon for my research was published in December 2010 by <strong>Dr</strong>. James Derr and colleagues entitled "Complete mitochondrial DNA sequence analysis of Bison bison and bison-cattle hybrids: function and phylogeny". It is attached to my declaration. Another key document concerning the nearest Hving relative of bison was published in December 2010 by Zhaofang Wang and colleagues entitled "Phylogcographical analyses of domestic and wild yaks based on mitochondrial DNA: new data and reappraisal". The synthe:sis of new data in these two papers and many others established that certain bison genetic variations are not hannless variants but instead deleterious mutations. 10. I conducted analysis of complete bison mitochondrial genumes 1n a comparative genomics context and established that a widespread bison mitochondrial genome, haplotype 6, carries substitutions (relative to haplotype 8) in both cytochrome b (V98A: the amino acid valine at position 98 in hap H is changed to alanine in hap 6) and ATP6 (I60N: isoleucine at position 60 in hap 8 is changed to asparagine in hap 6). Both variants in haplotype 6 are unambiguously deleterious according to numerous bioinfonnatic criteria and clinical observations in other species; together as a double mutation the strongly imply that these bison are affected by significant mitochondrial disease. 11. Since similar mutations in human and dog cause consistent clinical impairment of energy production from food (via mitochondrial oxidative phosphorylation). these bison are predicted significantly impaired in aerobic capacity, plausibly disrupting highly evolved cold toleran.ce, winter feeding behaviors, escape from predators and competition for breeding. 12. To estimate prevalence of diseased and non-diseased baplotypes at YNP, I used all available data as of January 15,2011. This consisted of two YNP complete mitochondrial genomes that speak: directly to disease or non-disease status but primarily of shorter mitochondrial control region sequences obtained by F. Gardipee for 151 YNP and 28 GINP bison with representative saItlpling from known geographical locations. These later sequences did not directly cover the two regions PAGE 04/ 05
02/14/2011 10:36 5206827080 PICTURE ROCKS HDWR of interest (cytochrome b and A TP6). However, the variety of controls and re-analysis described in my paper strongly indicate that the Gardipee data can be unambiguously mapped into disease or notdisease ha.plotype and these shorter sequences can thus serve as a reliable proxy for full data, as they routinely are throughout biomedical genetics. The Ga.rdipee thesis is attached to my declaration. 13. Below Ire-cast Gardipee's Table 3-1 into disease (hap6) and nondisease (hap8) coIwnns under the assumption that truncated microhaplotypes still provide a valid window into the status of the protein coding genes cytochrome b and A TP6. PAGE 05/06 V98A I60N V98V 1601 hap 6 hap 8 Park Herd Hayden (deleterious) (healthy) % Healthy YNP YNP Valley Lamar Valley 88 19 6 22 6% 54% YNP Mirror Plateau 10 6 38% GTNP Antelope Flats 20 0 0% GlNP WolfCreek 8 ° 0% 14. In some years the cull size has exceeded 1,000 animals and in one year the cull was approximately 1,400 individuals which may significantly exceed the total number of estimated hap 8 bison in the park (some 778 animals. assuming averaged YNP data above is roughly representative of current Park bison with total herd size taken as 3,500). However Park hison move about and herd numbers fluctuate; direct DNA testing of coralled animals prior to cull would be a more accurate way of measuring non-disease animals slated for cull. 15. Because the hap 8 (healthy) bison are disproportionally in the Northern herd of the Park according to the best available data (Gardipee), they may be over-represented in corralled animals near Gardiner destined for slaughter, disproportionally reducing the number of hap 8 animals. These data suggest 2011 culls could worsen
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Page 8 and 9: Below, analysis of complete bison m
Page 10 and 11: • The mutation rate in mitochondr
Page 12 and 13: possibility of mitochondrial dysfun
Page 14 and 15: arginine R100 provides an essential
Page 16 and 17: 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
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visualized, assessed for quality, a
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aligned with less than two out of t
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ears (Ursus americanus), and brown
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Figures: Figure 2-1. Flow diagram s
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Table 2-2. Amplification success (A
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Appendix 2-3. Allelic dropout (AD)
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strong fine scale genetic different
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fidelity to breeding areas may be h
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RFLP PCR amplification was carried
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etween the Lamar Valley and Hayden
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and Lamar Valley, in comparison to
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The differences in haplotype freque
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Figures: Figure 3-1. Approximate lo
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Literature Cited Allendorf, F.W., R
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Côté, S.D., F. Dallas, F. Marshal
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Luikart, G., S. Zundel, D. Rioux, C
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Tedford, S. Zimov, and A. Cooper. 2
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general approach using mtDNA marker
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2 K.C. Douglas et al. / Mitochondri
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mitochondrial genome is 16318-16323
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Bison Herd (bHap13 and bHap16), the
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spread distribution of bison prior
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Declaration Dr. Thomas H. Pringle - Buffalo Field Campaign

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