Tia Beavert

1
Tia Beavert

• Makah Potato Genomics Workshop
• 22 of June to 3 of July, 2003
• USDA/ARS
• National Science Foundation

2
Those Involved

• Charles Brown
• Cassandra Smith
• Hazel Denney
• Tia Beavert
• Neuee Ward
• David Culley
• Daniel Andrade

3
Our Purpose
Focus will be using genetic markers to determine
origin of the Ozette Potato, an important Makah
food and a possible Andean relict. Its
introduction is lost in history.
Makah
Yakama
4
My Purpose
My purpose of attending this workshop was to
learn how to trace the DNA of plants using the
potato as a basis, hopefully in the future using
it to trace the DNA origin of our natural foods
and medicines.
5
Potatoes, Potatoes, Potatoes...
There are many types of potatoes
6
Day One Education

• Discussions,

Explanations,
Preparation
In order to do, one must understand.
7
Lectures, Videos, and Activities
They always said hands on activities helped
students learn better
8
DNA
Deoxyribonucleic Acid
Our models of DNA look much better.
9
The basic concepts of DNA are grasped
10
More Potatoes...
11

• Grind leaf tissue in a pre-chilled mortar in
  liquid nitrogen to a fine powder.
• Transfer 100mg powder into an Eppendorf tube. Add
  700µl of fresh 2X CTAB buffer (and 2µl of
  ß-mercaptoethanol and vortex). Place in 65C
  water bath for 45 min, inverting tubes every 15
  min. Cool samples to RT for 2 min.
• Add 700µl of chloroformisoamyl alcohol (241) to
  each tube. Vortex briefly and gently to avoid
  shearing DNA, then turn over several times.
• Spin 5 min at 14,000 rpm in a microcentrifuge.
  Remove aqueous top layer and transfer to a new
  labeled Eppendorf tube. Be careful to avoid
  carrying over interphase material. Dispose
  chloroformisoamyl alcohol waste properly in a
  labeled waste container.
• Add 50µl of 10 CTAB (in 0.7 M NaCl), vortex
  gently, and mix thoroughly.
• Repeat steps 3 and 4.
• Add equal volume of cold (-20C) isopropanol
  (400-500µl) to each tube. Turn over several times
  and let tubes sit at 4C for 30 min (15 min at
  -20C or 5 min at 70C).
• Spin at 14,000 rpm for 20 min. Carefully pour off
  supernatant so as not to lose the DNA pellet
  turn over tubes to dry (1-2 min).
• Wash pellet with 1ml 70 EtOH (at least 1-3 min)
  and spin 30 min at 14,000 rpm. Carefully pour off
  EtOH, wash pellet in 1ml 95 EtOH, spin 30 min at
  14,000 rpm, and carefully pour off EtOH. Turn
  tubes upside down and air-dry overnight or for
  few minutes using a vaccum pump.
• Dissolve DNA in 150µl T10E1 or distilled H2O per
  sample, add 1-2µl 10mg/ml (DNAse-free)-RNAseA,
  vortex briefly and gently, and spin briefly.
  Incubate at 37C for 1 hr.
• DNA concentration is estimated in comparison with
  known concentration of lambda DNA in 1 agarose
  gel.
• Store at 4C, or at -20C for long-term storage.

Protocol
The process of DNA Extraction required the
process shown. It was more interesting than what
the words made it look.
12
DNA Lab Work
Day Two
Leaves from the potato plant were placed in micro
centrifuge tubes, frozen by liquid Nitrogen, and
crushed with kontes pestles.
(Dave shows us how it is done.)
13
Dont break the concentration!
14
More Lab Work...
Pipetting.
Freezing the leaves.
15
The Final Product looks like this
Cassandra Hazel
Me
16
Not Like This!!!
17
So...
18
In order to get a acceptable product, we had to
complete the process over.
19
Repeating the Process
The first process we completed did not turn out
as planned. The error effected the final
outcome, but the outcome of the second final
product is much better.
20
Over the weekend work...
Making a master mix
Loading the sequencing gel that separates the DNA
by size
Observing a part of the process
21
Other Presentations...
22
Nematodes
Dr. Hassan Mojtahedi
Nematodes are non-segmented, roundworms,
generally microscopic, usually wormlike,
free-living, or parasitic of plants, animals, and
man.
Some diseases nematodes give potatoes.
23
The Tobacco Rattle Virus causes corky ring spot
disease on potatoes.
Nematodes can cause disease in potatoes as well
as other plants
24
Fungi Water Molds
As Nik Grunwald talked
We listened.
25
Corn smut
Types of Fungi
Agaricus bisporus
This is the most commonly grown mushroom in the
United States, accounting for up to 90 of the
mushroom production here.
26
Soil borne Diseases
Presentation by George Vandemark
Seedling diseases
Crown Rots
Stand Decline
Winter Kill
27
George Vandemark explain the math behind
gene Replication and amplification.
28
Presented by Dr. Jim Crosslin
Viruses need a living cell or a host to survive
because they are not free living.
A
B
C
D
Development of invading M. chitwoodi in root (all
at 25 days post-inoculation. 60x).
Solanum tuberosum
Solanum fendleri.
Solanum hougasii.
Solanum bulbocastanum
29
Thank You!