Genetic Diversity among Imperata cylindrica

1. Genetic Diversity among Imperata cylindrica Josiah Raymer, Undergraduate Research Assistant Mack Thetford, Associate Professor West Florida Research and Education Center, Milton, FL Institute of Food and Agricultural Sciences University of Florida

2. Background InformationImperata cylindricaCogon Grass • Invasive weed • No reliable methods of control • Obligate outcrosser (can not self fertilize) • Lack of genetic diversity (clonal populations) • Red leaf variants are sold as an ornamental • Limited knowledge of genetic relationship

3. Imperata cylindricaCogon Grass Variety? Cultivar? Cultivar of a variety? Cranberry Grass Red Baron Grass Red Baron Cranberry Grass Imperata cylindrica rubra Imperata cylindrica ‘Red Baron’ Imperata cylindrica rubra ‘Red Baron’

6. Objectives • Develop a reliable method of extracting amplifiable DNA from both red and green leaf selections of Imperata • Determine if diversity exists among red leaf selections of Imperata

7. Objective 1 • Determine a reliable DNA extraction method • Evaluate the effectiveness of two alternative DNA extraction procedures compared to the standard CTAB procedure to obtain amplifiable DNA

8. Qiagen DNeasy® Extraction • DNeasy Plant Mini Kits • A commercially available Plant DNA extraction kit

9. Amplification • RAPD/PCR reactions • Replication of DNA fragments of specific size based on the primer used in the reaction • Gel Electrophoresis • Separation of DNA fragments of specific size using an electrical current

10. Amplification (Qiagen) • DNA from green leaf samples produced well amplified fragments DNA from red leaf samples did not produce amplified fragments

11. DNA Purification (red samples) • Centricon YM-100 ultrafilters • Membrane based filtration tubes

12. Results of Amplification UltrafilteredDNAisolatedfrom red leaf Imperata viaQiagen DNeasy kits, produced amplified fragments

13. Results • DNA of green leaf Imperata extracted using the Qiagen DNeasy kits produced amplifiable DNA • DNA of red leaf Imperata extracted using the Qiagen DNeasy kits required an extra ultrafiltration step before amplification occurred

14. Objective 2 • Is There Genetic Diversity Within Selections of Red Leaf Cogongrass? • Are Red Leaf Selections similar to Green Leaf Selections?

15. Samples • 29 Samples • 13 green leaf (UF Collection) • 16 red leaf (multiple industry sources)

16. Amplification • RAPD/PCR • 5 Primers • 5 Different fingerprints/sample • Horizontal gel electrophoresis • Digital Images

17. 1 Kb ladder 1 Kb ladder 1 Kb ladder A19 A09 A09 A19 A02 A02 A07 A07 B02 B02 Sample #9 Sample SB120 Gel Images Imperata cylindrica Cogon Grass

18. Gel Processing • Gel imaging software (Gel Comprar II) • 5 fingerprints for each sample were linked to form a composite data set • Dendogram was constructed • Principal component analysis • Jacknife comparison

19. Dendogram Imperata cylindrica Cogon Grass

20. Principal Component AnalysisImperata cylindrica Cogon Grass

21. Jackknife comparisonImperata cylindrica Cogon Grass

22. Conclusions Red and green leaf selections of Imperata cylindrica were separated into two distinct groups.

23. Conclusions Red leaf selections of Imperata cylindrica were further separated into two distinct groups.

24. Significance to Industry • Genetic diversity is present within populations of red and green leaf selections of cogongrass. • Potential exists for cross pollination between the different selections.