1. PCR to Distinguish Diploid and Triploid Forms of Butomus umbellatus
Amy Stiffarm, Elizabeth Rutledge, Ph.D Department of Life Sciences, Salish Kootenai College; Cellular Molecular Biology Laboratory, Pablo, MT

2. Introduction

3. Introduction: Proposed Test
Diploid chromosomes
Triploid chromosomes
SNPs
In the diploid form there are equal numbers of each allele of a SNP.
In a triploid there will be two copies of one allele and one copy of the other allele.

4. Introduction
By using the TaqMan Assay the diploid form can be distinguished from the triploid form.

5. Methods & Results: Obtain Sequence Data
Genbank data bases were search for Allium cepa (onion) and the Alismataceae family sequence data.
mRNA and partial coding sequence
between predicted exon boundaries (AGG and CGG)
greater than 100 base pairs in length.

6. Methods & Results: Obtain Sequence Data
PCR was used to test the primers sets along with the TOPO TA control reaction. Out of the total 23 primer sets designed for PCR 2 sets yielded amplicons.

7. Methods & Results: Obtain Sequence Data
Gel Electrophoresis was used to analyze PCR products.
750bp
>1000bp
~200bp
~ bp
Zinctrans_F&R primers
Endhydro_F&R primers
Control primers

8. Methods & Results: Obtain Sequence Data
Zinctrans_F&R PCR products were sent to the Murdock Sequencing Laboratory at the University of Montana in Missoula, MT.
Sequencing reaction results yielded a 185 base pair region. From two separate sequencing reactions a possible SNP was observed from the chromatograms.

9. Methods & Results: Transformation
TOP10F’ chemically competent cells were transfected with the plasmids using the TOPO TA Cloning reaction with PCR products onto LB plates containing 50ug/ml carbenicillin and incubated at 37°C overnight.
Individual colonies were grown in LB broth containing 50ug/ml carbenicillin and incubated at 37°C at 250rpm overnight.

10. Methods & Results: Insert Verification PCR
Plasmid DNA was isolated from 6 cultures. PCR was used to verify successful transformation.
Control template DNA
Zinctrans_F&R amplicon
Endhydro_F&R amplicon
Four out the six plasmids that were isolated contained inserts.
~200bp= Vector only.
~100bp=pUC19 Control

11. Conclusions
Three different regions of the genome have been amplified. It appears that binding of primers is random at this point.
Sequence data suggests possible SNP.
Four of six plasmids contain an insert. The area from the zinctrans_F&R that was sequenced wasn’t the area that was inserted into the vector for the samples analyzed thus far.

12. Future Directions
PCR reactions will be optimized by using gradient temperatures on the annealing temperature, using a time gradient on the final extension step, and by varying the amount of DNA used for the reaction.
Investigations of SNPs will continue until one is found in a single copy region of DNA.
Regions containing the possible SNP can be analyzed by High Resolution Melt (HRM)

13. References
Haynes and Les (2004). Nature Encyclopedia of Life Sciences. “Alismatales”.
Invitrogen TOPO TA Cloning User Manual (2006)
Mara Johnson, Peter Rice, Virgil Dupuis, Sue Ball (2008). Flowering Rush (Butomus umbellatus) in Flathead Lake and River: An Integrated Invasive Plant Management Project. Center for Invasive Plant Management.
National Center for Biotechnology Information.
U.S.D.A., Natural Resource Conservation Service.
U.S.G.S. Butomus umbellatus.

14. Acknowledgements
Funding: National Institutes of Health (NIH), NIGMS RISE Program R25-GMO76323
Murdock DNA Sequencing Lab, University of Montana, Missoula, MT
Joy Futrell and Brenda Grewell of USDA-ARS Exotic & Invasive Weeds Research Unit, University of California, Davis
J. Kirwin Werner, Virgil Dupuis, and Alvin Mitchell of Salish Kootenai College