Phylogenetic Analysis of 24 Brassica Species using SSR Markers

1. Phylogenetic Analysis of 24 Brassica Species using SSR Markers By: Chavon T. Graves Alabama A & M REU Program 7/25/08 Fisk University

2. 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).

3. Introduction Phylogenetic methods can be used for many purposes: • 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.

4. Objective: To determine the relatedness of cultivated Brassica species by utilizing: • 1.SDS-PAGE to look at the protein profile of 24 different species and genotypes of cultivated Brassica. • 2.SSR markers to screen the genome of 24 different species of cultivated Brassica.

5. Part I: 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 Tasks:

6. SSR Markers • 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

7. Part I: Protein: • Extraction • Analysis Of Brassica seeds…

8. 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

9. 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

10. Values of Protein Concentration

11. ….cont’d

12. 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

13. Protein Profile from Cultivated Brassica Napus M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 • M. Marker 5. Oscar 10. Talent 15. Calgene • Cyclone 6. Westar 11. Ida-Gold 16. Plainsman • Flint 7. Casino 12. Titan 17. Wotan • Falcon 8. Arctic 13. Viking • Glossy Bingo 9. Kronos 14. Maestro

14. ….cont’d M 18 19 20 212223 24 M. Marker 22. Blk-Leg Susc 18. Abilene 23. Bladur 19. Ceres 24. Banjo 20. Jetton 21. Rasmus

15. Blackleg Disease in Canola • 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

16. Part II: DNA: • Extraction Before: • Analysis Of cultivated Brassica leaves… After:

17. Procedure for DNA Extraction: • 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.

18. Making / Running the DNA Gel: • 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. • 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.

19. Only 9 germinated Caterpillars Heat Black leg? Harvesting Results

20. DNA Gel Profile M 1 2 3 4 5 6 7 8 9 M. Marker 3. Ida Gold 6. Abilene 1.Flint 4. Casino 7. Oscar 2. Westar 5. Rasmus 8. Falcon 9. Jetton

21. Water 10x hot start PCR buffer dNTP mix Primer 1 (forward) Primer 2 (Reverse) 25mM mgcl2 Taq DNA polymerase Template DNA Prepared right concentration of the mixture Set a program in the PCR machine with the right Tm(melting temp) Run the contents of the PCR products on agarose gel Take pictures PCR Analysis

22. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Results of SSR-Analysis M. Marker 3. Ida Gold 6. Abilene 1.Flint 4. Casino 7. Oscar 2. Westar 5. Rasmus 8. Falcon 9. Jetton

23. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Results of SSR-Analysis (cont’d) M. Marker 3. Ida Gold 6. Abilene 1.Flint 4. Casino 7. Oscar 2. Westar 5. Rasmus 8. Falcon 9. Jetton

24. Data Analysis (Scoring DNA bands) + = band present 0 = band absent

25. Data Analysis (Scoring DNA bands)

26. Conclusion for Part I: 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

27. Conclusions for Part II: • 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.

28. Future work • Calculate polymorphism • Construct a phylogenetic tree to determine how closely related the genotypes are.

29. Special Thanks to…. • Dr. Koffi Konan • Anthony Ananga • Dr. Ernst Cebert • Dr. Elica Moss • REU Family

30. References • 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.