Molecular assessment of translocation and management of an endangered subspecies of white-tailed deer
(Odocoileus virginianus)
Conservation Genetics
Matthew W. Hopken1*
Tod M. Lum3
Paul M. Meyers4
Antoinette J. Piaggio1
Author affiliations:
1
USDA/APHIS/WS National Wildlife Research Center, 4101 Laporte Ave. Fort Collins, CO 80521, USA.
2
Oregon Department of Fish and Wildlife, 2900 Northwest Stewart Parkway, Roseburg, OR 97471, USA
3
Julia Butler Hansen National Wildlife Refuge, U.S. Fish and Wildlife Service, P.O. Box 566, Cathlamet, WA
98612, USA.
*Corresponding Author contact info:
Email: Matt.W.Hopken@aphis.usda.gov
Phone: 970-266-6046
Fax: 970-266-6063
Appendix (electronic supplementary material)
DNA extraction procedure for whatman FTA blood cards
The Whatman FTA blood cards were extracted with the Qiagen DNeasy® Blood and Tissue Kit. A 3 mm diameter
circle was removed from a dried blood spot on the FTA card with a single-hole paper punch sterilized by dipping in
ethanol and flaming with a Bunsen burner. The paper circle was placed in a microfuge tube and incubated overnight
at 56ºC with 180 µl of Qiagen Buffer ATL. The following day, 20 µl of proteinase K and 200 µl of Qiagen Buffer
AL were added to the lysis solution and incubated at 70ºC for 10 min. We then added 210 µl of 100% ethanol and
transferred the lysate to Qiagen columns and followed the remaining steps from the manufacturer’s protocol for
animal tissue samples.
Mitochondrial DNA PCR conditions
Each 25 µL reaction contained 5.5 µL of proprietary Promega Buffer C, dNTPs (1 mM), primers (1 µM of
each), Gotaq Flexi DNA polymerase (Promega) (1 unit) and 1 to 2 µL of DNA extract. For samples with weak
amplification we used 2.5 µL 10X Amplitaq Gold buffer, 1.5 mM MgCl2, 1.0 mM of dNTPs, 1.0 unit of Amplitaq
Gold DNA Polymerase (Applied Biosystem; ABI) and 2.0 µL of DNA extract. PCR cycling conditions were initial
denaturation at 94ºC for 4 minutes; 32 cycles of 94ºC for 1 minute, annealing at 52°C for 40 seconds, extension at
70°C for 1 minute with 3 seconds added each cycle and a final extension of 72°C for 7 minutes. When Amplitaq was
used, initial denaturation was increased to 95°C for 10 minutes.
Table A1. PCR conditions for Odocoileus microsatellite loci used in this study. There were four multiplexes and conditions
varied depending on species and sample type.
Odocoileus virginanus TISSUE SAMPLES
Water
Buffer C
dNTP (2.5 mM)
Primers F/R
Promega taq
Panel A
3.01 (µl)
5.5
2
0.07 INRA011-10uM
0.09 R-10uM
0.21 P-10uM
Qiagen Multiplex kit
Promega taq
BSA (5 µl/ µg)
Total Mix
Template DNA
0.1-0.2
0.4
11.75
1
0.2
0.4-0.6
14.3
1
PCR Profiles:
95º 5 m // 1 cycle
95º 5 m // 1 cycle
Panel B
2.7 (µl)
6
2
0.4
0.4
0.1
0.4
Water
Buffer 2x
BM4208-10uM Primers F/R
BovPRL-10uM
BM6506-10uM
N-10uM
Total Mix
Template DNA
PCR Profiles:
PCR Profiles:
BL25-1uM
K-1uM
BM6348-1uM
Cervid1-1uM
ILSTS011-10uM
0.3
0.75
0.1
0.8
BM848-10uM
Q-1uM
D-10uM
OarFCB-1uM
11
1*
95º 15 m // 1 cycle
95º 15 m // 1 cycle
95º 15 s / 52º 30 s / 72º 1 m // 30-35 cycles 95º 15 s / 50º 30 s / 72º 1 m // 30 -37cycles
94º 30 s / 57º 90 s / 72º 1 m // 35 cycles 94º 30 s / 62º 90 s / 72º 1 m // 35 cycles
72º 45 m // 1 cycle
72º 45 m // 1 cycle
60º 30 m // 1 cycle
60º 30 m // 1 cycle
4º ∞
4º ∞
4º ∞
4º ∞
Panel A
1.2 (µl)
5
0.6 INRA011-1uM
0.1 R-10uM
0.2 P-10uM
8
2
*Panel D product needs to be diluted anywhere
from 1:2 to 1:10 before loading on Genetic
Analyzer
Qiagen Multiplex kit
Panel B
0 (µl)
6
0.5 BM4208-10uM
0.5 BovPRL-10uM
0.5 BM6506-10uM
0.5 N-10uM
10
2
Panel C
0 (µl)
6.5
0.5
0.5
0.15
0.8
0.8
12
2
Panel D
1 (µl)
6
BL25-1uM
K-1uM
BM6348-10uM
Cervid1-1uM
ILSTS011-10uM
0.8
0.3
0.3
0.1
10
2
O-10uM
BM848-10uM
Q-10uM
D-10uM
OarFCB-10uM
95º 15 m // 1 cycle
95º 15 m // 1 cycle
95º 15 m // 1 cycle
94º 30 s / 52º 90 s / 72º 1 m // 35 cycles
94º 30 s / 50º 90 s / 72º 1 m // 38 cycles
94º 30 s / 57º 90 s / 72º 1 m // 45 cycles 94º 30 s / 62º 90 s / 72º 1 m // 40 cycles
60º 30 m // 1 cycle
60º 30 m // 1 cycle
60º 30 m // 1 cycle
60º 30 m // 1 cycle
4º ∞
4º ∞
4º ∞
4º ∞
Panel B
2.7 (µl)
6
2
0.4
0.4
0.3
0.4
Panel C
1.15 (µl)
6.25
Odocoileus hemionus deer TISSUE SAMPLES
Promega taq
Panel A
Water
2.22 (µl)
Buffer C
5.5
dNTP (2.5 mM)
2
Primers
Primers F/R
0.07 INRA011-10uM
0.11 R-10uM
0.21 P-10uM
Promega taq
BSA (5 µl/ µg)
Total Mix
Template DNA
0.25
0.3
0.5
0.65
0.6
Panel D
0.85 (µl)
6.25
12
1
Odocoileus virginianus FTA BLOOD CARDS
Water
Buffer 2x
Primers F/R
Panel C
1.15 (µl)
6.25
0.1
0.4
11
1
95º 15 m // 1 cycle
Qiagen Multiplex kit
Water
Buffer 2x
Primers
BM4208-10uM Primers F/R
BovPRL-10uM
BM6506-10uM
N-10uM
0.2
0.6
14.5
1
0.25
0.3
0.5
0.65
0.6
Panel D
1.05 (µl)
6.25
Primers
BL25-1uM
K-1uM
BM6348-1uM
Cervid1-1uM
ILSTS011-10uM
12
1
0.35
0.6
0.1
0.8
Primers
BM848-10uM
Q-1uM
D-10uM
OarFCB-1uM
11
1
95º 5 m // 1 cycle
95º 5 m // 1 cycle
95º 15 m // 1 cycle
95º 15 s / 52º 30 s / 72º 1 m // 30 cycles
95º 15 s / 50º 30 s / 72º 1 m // 32 cycles
94º 30 s / 57º 90 s / 72º 1 m // 35 cycles 94º 30 s / 62º 90 s / 72º 1 m // 35 cycles
95º 15 m // 1 cycle
72º 45 m // 1 cycle
72º 45 m // 1 cycle
60º 30 m // 1 cycle
60º 30 m // 1 cycle
4º ∞
4º ∞
4º ∞
4º ∞
Table A2. Parameters for the model of
molecular evolution, TVM+I+G, that was
determined by jMODELTEST to have the best fit
to our data. This model was used to construct a
maximum likelihood phylogenetic tree.
Nucleotide Frequencies
A
C
G
T
0.318
0.120
0.215
0.347
Substitution rate matrix
AC
AG
AT
CT
GT
4260.035
153660.638
7046.718
153660.638
1.000
Prop.Inv
0.552
Alpha
0.313
Table A3. Haplotypes from Clades A and B that were found in O. virginanus in the Pacific Northwest. The haplotype
identifier from Figs 2 and 3, number of samples with each haplotype (n), the current subspecific designation, sampling
location and the state where the individual was collected, and GenBank accession numbers for haplotypes found in O.
virginianus. Accession numbers for all haplotypes in this study range from KP308220 to KP308271.
Haplotype
n
Subspecies
a
11
O. v. leucurus
LC/JBH
Sampling location
Oregon (Tenasillahe Island), Washington (M ainland)
State
Accession
KP308222
b
44
O. v. leucurus
DCOR
Oregon
KP308262
c
42
O. v. leucurus
LC/JBH
Oregon (M ainland, Tenasillahe Island)
KP308266
d
19
O. v. leucurus
LC/JBH
Oregon (M ainland), Washington (Puget Island)
KP308235
e
1
O. v. ochrorous
NWWTD
Oregon
KP308231
f
13
O. v. ochrorous
NWWTD
Oregon
KP308269
g
22
O. v. ochrorous
NWWTD
Oregon
KP308270
h
9
O. v. ochrorous
NWWTD
Oregon, Washington
KP308243
i
2
O. v. ochrorous
NWWTD
Washington
KP308254
j
3
O. v. ochrorous
IDWTD
Idaho
KP308257
k
1
O. v. ochrorous
NWWTD
Washington
KP308245
l
1
O. v. ochrorous
NWWTD
Oregon
KP308263
m
2
O. v. ochrorous
IDWTD, NWWTD
Idaho, Washington
KP308237
n
1
O. v. ochrorous
WYWTD
Wyoming
KP308230
o
2
O. v. ochrorous
IDWTD, WYWTD
Idaho, Wyoming
KP308241
p
1
O. v. ochrorous
NWWTD
Washington
KP308248
q
1
O. v. ochrorous
IDWTD
Idaho
KP308256
r
1
O. v. ochrorous
IDWTD
Idaho
KP308224
s
3
O. v. ochrorous
NWWTD
Oregon, Washington
KP308271
t
2
O. v. ochrorous
NWWTD
Oregon
KP308249
u
1
O. v. ochrorous
NWWTD
Washington
KP308264
v
2
O. v. ochrorous
IDWTD
Idaho
KP308221
w
9
O. v. ochrorous
IDWTD, NWWTD
Idaho, Oregon, Washington
KP308234
x
1
O. v. ochrorous
WYWTD
Wyoming
KP308225
y
8
O. v. ochrorous*
NWWTD
Oregon, Washington
KP308238
z
1
O. v. ochrorous
NWWTD
Washington
KP308267
*also shared with four O. h. hemionus