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An Intra and Inter Herd Genetic Study Using SNP Analysis of Mule Deer (Odocoileus Hemionus) in Washington State

Today in Colorado the mule deer population is estimated to be below 600,000 individuals. This is drastically less than the estimated peak population in the 1940s. Similarly California’s mule deer population has decreased to half the size it was in the 1950s. This same pattern continues throughout the mule deer’s native habitat; the western United States. With this population decline there is also the potential for a decline in genetic variation. Using Single Nucleotide Polymorphisms (SNP’s) our research team is looking at intra and inter herd diversity within a population around eastern Washington. Extracting DNA from blood samples using Qiagen Blood and Tissue Kit, we are sequencing DNA from the CO I gene and the mitochondrial D-loop DNA to look for single point mutations.

ABSTRACT

METHODS AND MATERIALS

Cycle SequencingForward and Reverse cycle sequencing master mix were prepared for both the D-Loop primers and CO I primers. Master Mix for the D-Loop primers was made to have a gradient. 7uL of master mix was made to be combined with a total of 3uL of water and Freeze and Squeeze template. CO I master mix made with the same gradient. Then the samples were ran through cycle sequencing (Table 3).

Cycle Sequencing Clean-upCycle Sequencing products were purified using the Cycle Sequencing clean-up protocol from the Automated DNA Sequencing chemistry guide from Applied Biosystems. Two modifications were made to maximize results. At the end of step number eight, the supernatants were aspirated carefully from the PCR tubes, and then the lids from the tubes were cut and the tubes were placed upside down into 1.5mL tubes and centrifuged at 1,000rpm’s for an extra two minutes. This was done to help remove any excess liquid missed with the pipettes. Next, the dry setting on the Thermal Cycler at 90 C was used to dry the samples again for two minutes rather than one.

Capillary Sequencing The samples were re-hydrated using HiDye Formamide and loaded into an Applied Biosystems 310 Genetic Analyzer and sequenced using 310 Data Collection Software Version 3.0.0.

METHODS AND MATERIALS CONTINUED CONCLUSIONS

Currently in our research we have made progress in some different areas. Using the CO I primers we have found a pseudo-gene in the genome. This pseudo-gene is a region of DNA that has a landing site for our primer that until this point was unknown. This primer was found in a gel from a PCR reaction when two different segments of DNA were amplified from the CO I primer (fig. one). One was the piece we had been trying to amplify and the other was the new pseudo-gene. We are now planning on sequencing on both of these DNA segments and publishing our findings. Amplifying the D-loop primers as shown in Figure 2, we were able to use the process of freeze and squeeze and successfully take the DNA into sequencing. How the sequence appears is shown in Figure 3. No conclusions can be drawn at this time as to the genetic differentiation of the mule deer, but we have proofed our methods and materials and now we will continue to extract DNA and retrieve sequence to increase our end value. This will give us enough results to draw a clear conclusion soon.

REFERENCES

Cook C. Rachel et. al. Revisions of Rump Fat and Body Scoring Indices for Deer, Elk, and Moose. Journal of Wildlife Management

Robinson S. Hugh et. al. To Twin or Not to Twin? Trade-off in Litter Size and Fawn Survival in Mule Deer. Journal of Mammalogy

Tollefson N. Tory et. al. Influence of Summer and Autumn Nutrition on Body Condition and Reproduction in Lactating Mule Deer. Journal of Wildlife Management

Shipley A. Lisa et. al. Validating Predictive Models of Nutritional Condition for Mule Deer. Research Article

Perez L. Elvia, Natural Selenium and Planted Forages: Effect on Mule Deer and Elk in Washington. Washington State University

Tollefson N. Tory, The Influence of Summer and Autumn Forage Quality on Body Condition and Reproduction of Lactating Mule Deer and Their Fawns (Odocoileus Hemionus). Washington State University

Latch K. Emily et. al. Species-wide phylogeography of North American mule deer (Odocoileus hemionus): cryptic glacial refugiaand postglacial recolonization. Molecular Ecology

AKNOWLEDGMENTS

Woodrow L. Myers, Department of Fish and Wildlife

James K. Ashlock, North Central High School

Randall S. James, North Central High School

Steve Fisk, North Central High School

New England BioLabs

Sample CollectionMule Deer blood samples were collected from multiple herds within Washington State. Tracking devices with specific frequency numbers were placed on multiple deer in the numerous herds. Periodically throughout the year, the deer were caught so they could be measured in size and so blood, hair, and feces could be collected. The samples and information collected from each deer were sorted by the frequency number on their tracking device and by herd. The blood samples were stored in K2EDTA at below freezing temperatures. In the lab, each frequency number representing each different deer was selected for further testing.

ExtractionsUsing the QUIAGEN DNA extraction kit (DNeasy 96 Blood & Tissue Kit 4 catalog number 69581) the Mule Deer DNA was extracted. We performed the procedure for non-nucleated blood (red blood cells) as opposed to nucleated (white blood cells) or cultured cells. We adjusted the volume of blood, PBS, and proteinase K to equal 220ul with 20ul of proteinase K, 75ul of blood, and 125ul of PBS.

PCRThe DNA was amplified using PCR (polymerase chain reaction) Master Mixes were made from primers specific to the Mule Deer’s mitochondrial D-loop DNA as well as CO I primers specific to another region of Mule Deer DNA (Table 1). 18uL of master mix (2mM MgSO4) was combined with 2uL of extracted template. The unamplified samples then underwent thermal cycling (Table 2).

Agarose Gel Electrophroesis and Imaging Each sample was then combined with 5uL of LB+ SYBR Gold (10X) to amplify the DNA fragments when ran through a 1.5% agarosegel. The gels were then imaged and captured by Kodak Imaging Device. The images were then analyzed to find DNA bands and the bands were then taken into the Freeze and Squeeze process.

Table 3: Cycle Sequencing Amplification Thermal Cycle ProfileAction Temperature Time (min)

Rapid Thermal Ramp 96.0 C 1:00

25 Cycles 96.0 C 0:1050.0 C 0:0560.0 C 4:00

Rapid Thermal Ramp 60.0 C 4:00

Rapid Thermal Ramp 04.0 C ∞

Hannah Roberts, Alyssa Barton, and Jacob EcklundNorth Central High School, Spokane, Washington

Table 2: PCR Amplification Thermal Cycle Profile Action Temperature Time (min)Hold 94.0 C 4:0035 Cycles 94.0 C 0:30

60.0 C 0:1072.0 C 0:40

Two Holds 72 4:00

Table 1: Primer Sequences Forward and ReversePrimer Sequence

D-Loop Forward 5’-TGT CAA CAT GCA TAT CCC GTC CCT-3’

D-Loop Reverse 5’-TTA GAC GGC CAT AGC TGA GTC CAA-3’

CO I Forward 5’-GGT GCT CCA GAT ATA GCA TTT-3’

CO I Reverse 5’-TCC TGC TGG ATC GAA GAAGGT-3’

RESULTS

GRAPHICS

Figure 1: CO I PCR results 

Figure 2: D‐Loop PCR results

Figure 3: DNA Sequence 

MULE DEER