Genetic Diversity among Genetic Diversity among Imperata cylindricaImperata cylindrica

Josiah Raymer,Josiah Raymer,Undergraduate Research AssistantUndergraduate Research Assistant

Mack Thetford,Mack Thetford,Associate ProfessorAssociate Professor

West Florida Research and Education Center, Milton, FLWest Florida Research and Education Center, Milton, FL

Institute of Food and Agricultural SciencesInstitute of Food and Agricultural Sciences

University of FloridaUniversity of Florida

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

Background Information Imperata cylindricaImperata cylindrica Cogon Grass

Cranberry Grass

Red Baron Grass

Red Baron Cranberry Grass

Imperata cylindrica rubra

Imperata cylindrica ‘Red Baron’

Imperata cylindrica rubra ‘Red Baron’

Imperata cylindricaImperata cylindrica Cogon Grass Variety?Variety? Cultivar?Cultivar? Cultivar of a variety?Cultivar of a variety?

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

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

Qiagen DNeasy® Extraction

DNeasy Plant Mini Kits

A commercially available Plant DNA extraction kit

Amplification

RAPD/PCR reactionsReplication of DNA fragments of specific size based on the primer used in the reaction

Gel ElectrophoresisSeparation of DNA fragments of specific size using an electrical current

Amplification (Qiagen)

DNA from green leaf samples produced well amplified fragments

DNA from red leaf samples did not produce amplified fragments

DNA Purification (red samples)

Centricon

YM-100 ultrafiltersMembrane based filtration tubes

Results of Amplification

Ultrafiltered DNA isolated from red leaf Imperata via Qiagen DNeasy kits, produced amplified fragments

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

Objective 2

Is There Genetic Diversity Within Selections of Red Leaf Cogongrass?

Are Red Leaf Selections similar to Green Leaf Selections?

Samples

29 Samples13 green leaf (UF Collection)

16 red leaf (multiple industry sources)

Amplification

RAPD/PCR5 Primers

5 Different fingerprints/sample

Horizontal gel electrophoresis

Digital Images

Gel Images Imperata cylindrica Imperata cylindrica Cogon Grass

1 K

b la

dder

1 K

b la

dder

1 K

b la

dder

A02

A07

A09

A19

B02

A02

A07

A09

A19

B02

Sample #9 Sample SB120

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

DendogramImperata cylindrica Imperata cylindrica Cogon Grass

Principal Component AnalysisImperata cylindrica Imperata cylindrica Cogon Grass

Jackknife comparisonImperata cylindrica Imperata cylindrica Cogon Grass

Results of jackknife comparison of red and green leaf samples of Imperata cylindrica.

Actual Sample

Morphology

Predicted Sample Morphology

(%)

Green Leaf Red Leaf

Green Leaf 85 15

Red Leaf 0 100

Conclusions

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

Conclusions

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

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.