t

1
t
NSF Potato Genome Project Outreach Program
I. Background
The NSF Potato Genome Project is committed to
promoting education in the sciences. Three
genomics programs have been established to
introduce high school and college students, as
well as teachers and the general public, to the
importance of plant biodiversity and the rapidly
emerging field of genomic research. Heritage
College is establishing a program in which
students will study the Ozette potato for
marketable characteristics, and develop a plan
for an agricultural business. Working together
with the NSF Potato Genome Project, students and
biology teachers of Makah Nation will follow a
genomic trail leading to the origin of the Makah
potato. The Baker Lab summer genomics program, in
collaboration with the University of California
Botanical Garden and The Institute for Genomic
Research (TIGR), gives two El Cerrito high school
students from ethnically diverse backgrounds the
opportunity to design and conduct a genomics
experiment. Their goal is to produce genetic
markers,which will aid the Makah Nation in
identifying the origin of the Ozette Potato.
III. The Institute for Genomic Research (TIGR)
II. UC Berkeley Botanical Garden (UCBG)
UC Berkeley Botanical Garden
The Institute for Genomic Research (TIGR)
During our second week in the program we traveled
to Rockville, Maryland for genomic and
bioinformatic training at TIGRs Genomic Boot
Camp. This program provided us with information
about the future of genomic research, and gave us
the opportunity to practice DNA extraction
experiments using our own cheek cells. After we
completed the two-day course, we learned how to
data mine TIGRs Expressed Sequence Tag (EST)
database for microsatellite sequences. We were
able to design and use primers based on our
findings, revealing microsatellite sequences in a
variety of Solanaceous plants.
Our first week of the program was spent at the
University of California Botanical Gardens
(UCBG), where we learned to identify specific
characteristics of the Solanaceae family and the
Solanum (potato) genus. Working with the Crops
of the World garden coordinator, Lauri Twitchell,
we learned horticultural techniques such as
transplanting and creating sun shields. After we
planted a variety of wild and heirloom potato
species, we researched their significance and
created interpretive signage for the garden.
Upon public evaluation, we revised the signs for
permanent installation. Throughout the program
we returned to the UCBG to maintain our plants by
weeding and watering, and in September we will
return to harvest our potatoes.
Tara and Tomas load samples of DNA for sequencing.
Tara and Tomas carefully transplant wild potato
species into larger pots for distribution to
local elementary and middle school biodiversity
gardens.
Polymerase Chain Reaction (PCR)
PCR is a process by which an enzyme, Taq
polymerase, is used to make many identical copies
of a specific DNA sequence. DNA, loading dye,
buffers, taq enzyme and primers that anneal to
nucleotides flanking the DNA sequence of
interest, are loaded into a thermal controller
where they are exposed to temperatures optimal
for the primers and taq enzymes to synthesize DNA
fragments. After several cycles, a product
consisting of the amplified DNA fragment is
produced. Prior to PCR development, scientists
underwent long processes that produced few, if
any, DNA fragments. Due to PCRs ability to
produce many sequence copies using small amounts
of DNA, it has enabled scientists to
significantly accelerate their work. As a result,
PCR has helped define evolutionary relationships
among species, prevent people from being falsely
accused of crimes and provide large samples
necessary for medical research.
Willem Rensink, a Staff Scientist in the Buell
Lab, explains the effects of hormones on wild
potato plants.
NSF Potato Genome Project PI Barbara Baker works
with Tomas to transplant Ozette potatoes into
larger pots for transfer into the Crops of the
World Garden.
Joe Hsiao, a Research Associate in the Buell Lab,
shows Tara and Tomas how to load DNA samples into
their gels.
The Ozette potato sign created for the crops of
the world garden.
Microsatellites
Microsatellites are repetitive DNA sequences that
are likely to differ from species to species.
For criminal investigations or paternity suits,
thirteen microsatellite DNA sequences are
compared due to their variability among different
people. Scientists carefully analyze the DNA
samples, looking for similarities in
microsatellite sequences. Because
microsatellites are specific for each individual,
scientists can positively identify a suspect
within a few percentage points. Although
microsatellites vary among individuals, families
and closely related species often have similar
sequences that allow researchers to determine
evolutionary and familial relationships between
different species.
Tara, Tomas, Meghan Flanagan, Robin Buell, Willem
Rensink and Barbara Baker visit the National
Science Foundation building in Arlington,
Virginia.
Tara and Tomas plant wild potato species in the
Crops of the World Garden.
Lauri Twitchell shows Tara and Tomas how to set
up a sun shield for the young plants.
IV. Baker Lab
Microsatellites in Wild Solanum Species
Wild Potato Microsatellites
Introduction
The goal of our experiment was to look at
variable microsatellite sequences in seven
different wild potato species to determine their
genetic similarity. In addition, we wanted to
provide our collaborators in the Makah Nation
with genetic markers that will help them
determine the origin of the culturally
significant Ozette potato.
PI Barbara Baker shows Tara and Tomas the correct
protocol for working with cryogenic liquid
nitrogen.
Procedure
At TIGR, we designed primers, sequences of DNA
that surround a desired microsatellite region, to
compare seven wild potato species. To extract
the genomic DNA necessary for the experiment, we
harvested and ground young leaf tissue for
Polymerase Chain Reaction (PCR) DNA
amplification. We used four primers to isolate
microsatellite sequences specific to each potato
species. To determine which microsatellites
existed in each species, we ran our PCR products
on a gel that separates different sized DNA
fragments. We visualized the gel using UV light
and analyzed our results.
Results
Through UV visualization, we concluded that
Solanum hougasii and Solanum jamesii contain the
microsatellite (T)11, while Solanum demissum,
Solanum fendleri and Solanum sucrense have the
microsatellite (AT)7. Although none of the
species contain (A)11, (AAT)5 was found in all
species except Solanum sucrense and Solanum
bulbocastanum.
Gel Electrophoresis Results
Tomas and Tara grind tissue for their experiment
in the Baker Lab
Gel One
Gel Two
The gels to the right display our PCR
microsatellite products. We analyzed the gels
stained with Ethidium Bromide, which was mixed
with the products and can be visualized by UV
light. We compared the band migrations to a 100
base pair ladder that we loaded next to our DNA.
The bands underneath the wells represent our
desired microsatellite or primer. The potato
species that showed a band at 280kb contained the
desired microsatellite sequence.
Conclusion
Our experiment revealed that not all of the
microsatellites could be found in each wild
potato species, which means that each species has
its own unique genetic composition. This is an
important conclusion because it stresses the
importance of maintaining genetic diversity.
Unique wild potato genes can be introduced into
cultivated potatoes to enhance flavor and color,
or confer resistance to specific diseases. Thus,
our experiments support maintenance of
biodiversity around the globe.
Tara and Tomas discuss why old and disease
infected leaves are inappropriate for use in a
DNA extraction experiment.
VI. Acknowledgements
V. References
We would like to extend a special thanks to The
Institute for Genomic Research, especially Robin
Buell, Joe Hsiao, Jia Liu and Willem Rensink. We
would also like to thank the National Science
Foundation Assistant Director of Biological
Sciences, Mary Clutter, and Program Directors
Machi Dilworth, Jane Silverthorne and Anne
Sylvester. The UC Berkeley Botanical Garden
staff was an instrumental component of the
project, particularly Holly Forbes, Christine
Manoux, Lauri Twitchell and Jennifer White. Our
experience in the Baker Lab was made possible by
Barbara Baker, Meghan Flanagan, Maria Rosa
Marano, and XiaoXue Wang.
NSF Potato Genome Project http//potatogenome.org/
nsf3/ NSF Potato Genome Project Outreach
Program http//workshop.potatogenome.org/ TIGR htt
p//www.tigr.org/tdb/potato
UC Berkeley Botanical Gardens http//www.mip.berke
ley.edu/garden/ National Science
Foundation http//nsf.gov PCR Websites http//ww.f
aseb.org/opar/bloodsupply/pcr.html