Declaration Dr. Thomas H. Pringle - Buffalo Field Campaign

RFLP
PCR amplification was carried out as described above and digested with Ssp1,
which cuts haplotype 8, resulting in two fragments (372 bp and 98 bp in length).
Restriction digests were conducted in 20 μl volumes consisting of 11.3 μl sterile HPLC
water, 2 μl RE 10X buffer, 0.2 μl acetylated BSA (10μg/ μl), and 5 μl PCR product and
incubated at 37ºC for four hours. Digested products were run out on 2% agarose gels for
two hours. Gels were stained with ethidium bromide solution, and visualized using a
Hitachi FMBIOII scanner. We used 16 samples from YNP 2006 previously identified as
either haplotype 6 or 8 through sequencing as controls to test the accuracy of our RFLP
analysis. A set of 12 YNP 2005 samples identified as haplotype 6 were re-screened to
evaluate whether failure to digest could result in erroneous haplotype identification. No
haplotype identification errors were detected, thus validating the accuracy of the RFLP
analysis.
Data analysis
The combined results of sequencing and RFLP analysis were used to determine
the frequency and distribution of these haplotypes among the GYA bison populations,
and determine FST values. Significance of FST values were tested by contingency chi-
square analyses for comparisons among YNP breeding groups, and between parks.
RESULTS
Sequencing revealed two mtDNA control region haplotypes, amplified from
120 GYA bison fecal samples from the 2006 breeding season. These haplotypes matched
the first 408 bp of haplotypes 6 and 8 previously defined by Ward et al. (1999). RFLP
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