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489699.610008006004004
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Determining Artemisia tridentata Ploidy Number and the
Relationship to Branch Structure and Sage-Grouse Nesting
Mark Mendoza1,5
, Doug Redelman2,6
, David Shintani1
, Barry Perryman3
, and Christie Howard1,5
1
Department of Biochemistry and Molecular Biology; University of Nevada, Reno, 2
Physiology and Cell Biology, 3
Department of Agriculture, Nutrition
and Veterinary Sciences, 5
10,000 Hour Undergraduate Training Lab, 6
Nevada Cytometry Center
• Sage-grouse population rates have declined due to destruction
of sagebrush habitats. Protection of habitat is now under
consideration through the Endangered Species Act.
• Sage-grouse prefer to nest in U-shape branching that is found
in Big Sagebrush (Artemisia tridentata ssp. tridentata).
• Big Sagebrush stands that was burned in major fires across
Nevada have been restored, but in some regions they have
regrown with branching patterns that are V-shaped rather than
U-shaped.
• It is known that genome doubling can occur in Big Sagebrush.
• Is it possible that there is a factor that is causing Big
Sagebrush to have V-shape branching patterns instead
of U-shape patterning?
• The focus of this study is to determine whether there is
a relationship between ploidy number and branching
patterns in A. tridentata ssp. tridentata.
Introduction
Methods
Results Discussion
Future Directions
•Make protoplasts using enzymes such as cellulase and other
cell wall degrading enzymes to remove the cell walls.
•Use the protoplast samples to isolate nuclei.
References
Arumuganathan, K., & Earle, E. (1991). Estimation of nuclear DNA content ofArumuganathan, K., & Earle, E. (1991). Estimation of nuclear DNA content of
plants by flow cytometry.plants by flow cytometry. Plant Molecular Biology ReportePlant Molecular Biology Reporter,9(3), 229-41.r,9(3), 229-41.
Bennett M., Smith J. (1976). Nuclear DNA amounts in angiosperms.Bennett M., Smith J. (1976). Nuclear DNA amounts in angiosperms.
Philosophical Transactions of the Royal Society of LondonPhilosophical Transactions of the Royal Society of London B 274: 227-274.B 274: 227-274.
Bureau of Land Management. About the Greater Sage-Grouse. (2013, MarchBureau of Land Management. About the Greater Sage-Grouse. (2013, March
3). Retrieved August 11, 2014, from3). Retrieved August 11, 2014, from
http://www.blm.gov/wo/st/en/prog/more/sagegrouse/conservation.htmlhttp://www.blm.gov/wo/st/en/prog/more/sagegrouse/conservation.html
Garcia, S., Sanz, M., Garnatje, T., Kreitschitz, A., Mcarthur, E., & Vallès, J.Garcia, S., Sanz, M., Garnatje, T., Kreitschitz, A., Mcarthur, E., & Vallès, J.
(2004). Variation of DNA amount in 47 populations of the subtribe(2004). Variation of DNA amount in 47 populations of the subtribe
Artemisiinae and related taxa (Asteraceae, Anthemideae): Karyological,Artemisiinae and related taxa (Asteraceae, Anthemideae): Karyological,
ecological, and systematic implications.ecological, and systematic implications. GenomeGenome, 47, 1004-1014., 47, 1004-1014.
Richardson, B., Page, J., Bajgain, P., Sanderson, S., & Udall, J. (2012). DeepRichardson, B., Page, J., Bajgain, P., Sanderson, S., & Udall, J. (2012). Deep
sequencing of amplicons reveals widespread intraspecific hybridization andsequencing of amplicons reveals widespread intraspecific hybridization and
multiple origins of polyploidy in big sagebrush (Artemisia tridentata;multiple origins of polyploidy in big sagebrush (Artemisia tridentata;
Asteraceae).Asteraceae). American Journal of BotanyAmerican Journal of Botany, 99(12), 1962-1975., 99(12), 1962-1975.
Objective
Collect sagebrush leaf
tissue samples.
Ploidy Analysis
• A. tridentata ssp. tridentata (Big Sagebrush) has been
previously reported as having nuclear DNA content (C value)
of 4.09pg (Bennett) for diploid species.
• The flow cytometry results revealed that Big Sagebrush in our
study was well below the reported DNA content with the C
value at 1.3 pg.
• One possible explanation is that cell wall material is binding
to chloroplasts and nuclei giving false readings. The second
peak my actually be the G0/G1 peak with a value of ~ 4pg.
Slice samples and
suspend in buffer
with staining solution
propidium iodide (PI).
Run samples through flow
cytometer to detect nuclei.
Ploidy Analysis:
Figure 1. Optimization of Nuclei Isolation Procedure. Measurement of
relative fluorescence of nuclei for estimation of DNA content. A-F represent
chicken cell, trout erythrocyte, and trout triploid erythrocyte positive controls for
standard curve (Fig 2 below). G-J are nuclei isolated from tomato and squash
to confirm that the nuclei preparation was successful. K-L is the unknown
sagebrush sample.
Figure 2. Putative G0/G1 peak is migrating lower than expected
for Sagebrush nuclei. Flow cytometry results of Nuclear DNA.
Sagebrush result is 1.3pg /cell. This is less than expected for
sagebrush which should be approximately 4pg/cell. Controls used –
Chicken Erythrocyte Nuclei (CEN), Trout Erythrocyte Nuclei (TEN),
and Triploid Trout Nuclei (TTN).
Chicken Erythrocyte Nuclei
(CEN) Singlets, Triploid
Trout Nuclei, and Trout
Erythrocyte Nuclei (TEN)
were used as internal
standards.
Filter homogenate through nylon
mesh into microcentrifuge tube.
Centrifuge at high speed
(15,000 rpm) for 15 to 20
seconds and discard
supernatant.
Resuspend pellet in
buffer solution with
RNAse.
Incubate for 15
minutes at 37 C.
Suspension of nuclei from
plants with known DNA
content used as internal
standards.
Compare light scatter properties
with known standards to
determine nuclear DNA content.
CEN (control)
A
C
E
JI
G H
F
D
B
K L
G0/G1?