Transcript Slide 1

Phylogenetic Analysis
of 24 Brassica Species
using SSR Markers
By: Chavon T. Graves
Alabama A & M REU Program
7/25/08
Fisk University
What is Canola?
•A type of edible oil derived from plants
initially bred in Canada by Keith Downey
and Baldur Stefansson in the 1970s
•Brassica napus and Brassica rapa
(Mustard family)
•Many have been developed for food;
turnips, cabbage, brussel sprouts,
cauliflower, broccoli, mustard seed,
oilseed (rape).
Introduction
Phylogenetic methods can be used for many purposes:
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1. Analysis of morphological and several kinds of molecular data.
2. Comparisons of more than two gene sequences.
3. Analysis of gene families, including functional predictions.
4. Estimation of evolutionary relationships among organisms.
Objective:
To determine the relatedness of cultivated
Brassica species by utilizing:
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1.SDS-PAGE to look at the protein profile of
24 different species and genotypes of
cultivated Brassica.
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2.SSR markers to screen the genome of 24
different species of cultivated Brassica.
Tasks:
Part I:
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Protein Extraction from
Seeds
SDS-PAGE Analysis (Profile
of all Proteins)
Take Gel Pictures of Protein
Profiles
Data Analysis
Part II:
 Plant Canola seeds (24
genotypes for analysis)
 Harvest Leaves
 DNA Extraction
 DNA Electrophoresis (Check
Purity of DNA)
 Take Gel Pictures of DNA
Profiles
 Screening (PCR using SSR
Markers)
 Data Analysis
SSR Markers
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Simple Sequence Repeats (SSRs) or Microsatellites
Polymorphic loci present in nuclear and organellar DNA that
consist of repeating units of 1-6 base pairs in length.
Typically neutral, co-dominant and are used as molecular
markers which have wide-ranging applications in the field of
genetics, including kinship and population studies.
Can also be used to study gene dosage (looking for
duplications or deletions of a particular genetic region).
Microsatellites can be amplified for identification by the PCR
process
Part I:
Protein:
Extraction
Analysis
Of Brassica seeds…
Protein Extraction Procedure for Canola
Seeds
1. Weigh seeds (0.2g)
2. Grind in 0.5 M Tris HCl Buffer pH8 using 2mL (2000µL).
3. Pour into beaker. Label beaker and place Parafilm over.
4. Place in shaker for 2 hrs at medium speed (b/w 215-220)
5. Transfer to micro centrifuge (2mL)
6. Centrifuge at 3000 rpm for 5 min to remove particles
7. Transfer to micro centrifuge
8. Centrifuge at 13,000 rpm for 15 min
9. Transfer supernatant to micro centrifuge
10. Store at -20°C
Finding Protein Concentration in Canola
Seeds
1. Shake each micro centrifuge of extracted protein
using the vortex machine
2. Combine 1500 µL Coomassie Plus Protein Assay
Reagent and 50 µL of the extracted protein
3. Shake each mixture one by one
4. Allow to settle for 10 min
5. Read on spectrophotometer at 595 absorbance
6. Record readings
Values of Protein Concentration
Table 1.1
Species Name
Protein Conc. (µg/mL)
Cyclone
2431.6
Flint
2641.6
Falcon
2489.1
Glossy Bingo
2711.6
Oscar
2431.6
Westar
3164.1
Casino
2641.6
Arctic
2574.1
Kronos
2641.6
Talent
3326.6
Ida Gold
2899.1
Titan
2641.6
….cont’d
Species Name
Protein Conc. (µg/mL)
Viking
2469.1
Maestro
2711.6
Calgene
2469.1
Plainsman
2616.6
Wotan
2574.1
Abilene
2594.1
Ceres
2551.6
Jetton
2641.6
Rasmus
2686.6
Blk-leg Susc
2789.1
Baldur
3514.1
Banjo
3514.1
SDS Page (Sodium Dodecyl Sulfate
Polyacrylamide) Gel Electrophoresis
Objective: Run protein profile of Canola seeds
1. Making running buffer of 1X Tris Glycine SDS
2. Denature Protein
3. Load Protein
4. Let run at 200V for 1 hour
5. Stain gel for 15 min
6. De-stain for 30 min (2x)
7. Take picture
8. Store at -4 °C
Protein Profile from Cultivated
Brassica Napus
M 1
2
M. Marker
1. Cyclone
2. Flint
3. Falcon
4. Glossy Bingo
3 4 5 6 7
5. Oscar
6. Westar
7. Casino
8. Arctic
9. Kronos
8
9 10 11 12 13 14 15 16 17
10. Talent
11. Ida-Gold
12. Titan
13. Viking
14. Maestro
15. Calgene
16. Plainsman
17. Wotan
….cont’d
M
M. Marker
18. Abilene
19. Ceres
20. Jetton
21. Rasmus
18
19
20
21
22. Blk-Leg Susc
23. Bladur
24. Banjo
22
23
24
Blackleg Disease in Canola
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Blackleg is a serious disease of canola that
can result in significant yield loss in
susceptible varieties
It is caused by the fungus Leptosphaeria
maculans
Part II:
DNA:
Extraction
Before
Analysis
Of cultivated Brassica
leaves…
After
:
:
Procedure for DNA Extraction:
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Gather leaves.
Grind leaves with liquid Nitrogen.
Add 400 µL of Buffer AP1 and 4 µL of RNase soln to 1g of lysate.
Incubate mixture for 10 min in hot water bath of 65°C (mix
occasionally).
Add 130 µL of Buffer AP2 to lysate and incubate for 5 min on ice.
Centrifuge for 2 min at 14,000 rpm.
Add buffer AP3/E to lysate and mix by pipetting, and centrifuge for 1
min at 8,000 rpm. (Repeat)
Add 500 µL of buffer AW and centrifuge for 2 min at 14,000 rpm.
Pipette 100 µL of buffer AE and incubate for 5 min at room temp.
Centrifuge for 1 min at 8,000 rpm. (Repeat)
Store at -20° C.
Making / Running the DNA Gel:
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Combine 100 mL of 1X TAE Buffer, 0.5g of
Ethidium Bromide and 0.8g Agarose. Place
mixture in microwave for 2 min, then let cool.
Pour mixture in gel tray and let solidify for 45
min.
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Place 10 µL of the extracted DNA and 4 µL of
dye into each well. Fill gel tray with 1L of 1X
TAE Buffer. Let run for 2 hours.
Harvesting Results
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Only 9 germinated
Caterpillars
Heat
Black leg?
DNA Gel Profile
M
1
M. Marker
1. Flint
2. Westar
2
3
4
5
3. Ida Gold
4. Casino
5. Rasmus
6
7
8
6. Abilene
7. Oscar
8. Falcon
9. Jetton
9
PCR Analysis
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Water
10x hot start PCR buffer
dNTP mix
Primer 1 (forward)
Primer 2 (Reverse)
25mM mgcl2
Taq DNA polymerase
Template DNA
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Prepared right concentration
of the mixture
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Set a program in the PCR
machine with the right
Tm(melting temp)
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Run the contents of the PCR
products on agarose gel
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Take pictures
Results of SSR-Analysis
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9
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3
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9
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9
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9
1
M. Marker
1. Flint
2. Westar
3. Ida Gold 6. Abilene
4. Casino 7. Oscar
5. Rasmus 8. Falcon
9. Jetton
2
3
4
5
6
7
8
9
Results of SSR-Analysis (cont’d)
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9
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9
M. Marker
1. Flint
2. Westar
3. Ida Gold 6. Abilene
4. Casino 7. Oscar
5. Rasmus 8. Falcon
9. Jetton
1
2
3
4
5
6
7
8
9
Data Analysis (Scoring DNA bands)
+ = band present
0 = band absent
Data Analysis (Scoring DNA bands)
Conclusion for Part I:
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Most of the protein profiles appeared to be very similar
with the exceptions of:
Ida Gold
Cyclone
Plainsman
However some seemed to more closely related to each
other than others:
Abilene & Jetton
Ceres & Rasmus
Blk-leg Susc, Bladur, & Banjo
Conclusions for Part II:
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The SSR Markers proved to be very useful
in determining the variability of specific
regions of gene sequences in the Brassica
species (polymorphism).
I was able to determine if a specific band
was present, thereby signaling a genomic
relationship.
Future work
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Calculate polymorphism
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Construct a phylogenetic tree to determine
how closely related the genotypes are.
Special Thanks to….
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Dr. Koffi Konan
Anthony Ananga
Dr. Ernst Cebert
Dr. Elica Moss
REU Family
References
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www.academicjournals.org. African Journal of Biotechnology
Vol. 5. “RAPD Markers associated with resistance to blackleg
disease in Brassica Species.” 16 Nov. 2006.
Journal Agricultural Food Chemistry. “Protein Precipitating
Capacity of Crude Canola Tannins: Effect of pH, Tannin, and
Protein Concentrations” 20 May 1996.
Johnson, Duane. Crop Series. “Rapeseed/Canola
Production.” No. 0.110.