Jesse Migliori and Dr. Loreen Woolstenhulme, Botany and Range Science

Introduction

Microsatellites, or simple sequence repeats (SSRs) are genetic markers found throughout the genome. Usually consisting of short tandem repeats of 2-6 bases, SSRs offer molecular data from distinguishable loci. Because of their high mutation rates and high allelic diversity, microsatellites are ideal for genetic population studies, helping to establish relationships of single plants to the population or mating patterns within a population. Biologists and conservationists can take the information from the SSRs and develop a set of genetic fingerprints. The information provided by these fingerprints helps in the understanding of the plants genetic makeup and mating frequencies, ultimately leading to management strategies for endangered species. The focus of my research was in the development of microsatellites found in the genome of Kachina Daisy, Erigeron kachinensis.

An endemic plant to southeastern Utah and the Colorado plateau, the United States Forest and Wildlife Service declared the Kachina Daisy a threatened plant species. Due to small isolated populations, the resistance, fitness and genetic stock of these populations are reduced. Consequently, ovarian abortions, unmasking of lethal genes, genetic drift and mutations increase. Because the species’ habitat is restricted to seeps and alcoves along canyon walls, populations are being threatened by the increasing number of recreation enthusiasts who seek after these shady alcoves for hiking and resting. By building a database of microsatellites, the genetic flow can be accurately estimated among hanging garden species. Databases will also provide information for pollen movement among populations as well as the amount of inbreeding occurring in these populations.

Experimental

The lambda phage vectors were prepared according to the Zap Express Predigested Vector kit (Stratagene: La Jolla, Ca.). Tetracycline plates were used in growing the Ecoli colonies to reduce bacterial contamination. Using a sterile loop, a single colony of cells were transferred from the TET plate to a solution of 4 mL of LB-broth, 40μL of 0.1M MgSO4, and 40μL of a 4% Maltose solution. The cells were then incubated in solution for about 5 hours under perturbation. The process was then repeated by placing 40μL of the stock solution in a fresh broth solution and incubated for a couple more hours. The cell count was determined by an optical density (OD) measurement. The targeted percent absorbance for a 1mL cell was 0.500 OD at 600nm. The solution was partitioned into 250μL aliquots to which 50μL of the DNA library was added and incubated for 20 min. Culture tubes were prepared with top agar and the Ecoli, with the inserted DNA, combined and pour onto agar plates as bacterial lawns. The plates were allowed to grow over night.

Results and Conclusion

The experimental design was to take an existing DNA library generated from greenhouse-cultivated control specimens, consisting of predigested fragments of 300-600 nucleotides. The nucleotides would be isolated and cloned into a lambda phage vector (Zap Express Predigested Vector Kit, Stratagene: La Jolla, CA). The vectors will then be screened for SSRs using dinucleotide repeats, (CA)15 and (CT)15, and poly-(ATT) probes. These primary screens would be amplified and the DNA visualized on an agarose gel. A southern blot technique would verify preliminary screening by reducing false positives. For amplifications that tested positive for secondary screening, the remaining amplified DNA would be purified using Geneclean (Qbiogene: Carlsbad, CA) and sequenced using the BigDye terminator cycle sequencing reaction (PE Biosystems: Foster City, CA). The sequenced DNA would then be sent to the BYU Sequencing Center to be analyzed. From the analyzed data, primers will be designed to match sequences of clones for 15-20 highly variable microsatellite loci. Conditions for PCR will then be optimized for these variable loci.

Unfortunately, primers for the Kachina daisy were not developed. Isolation and cloning of the DNA vectors impeded the progress of the experiment. Even though the final goal of the experiment was not reached, I am fully confident that the experiment would have been successful once the DNA vectors were made. Modifications to the provided instructions were also made; changing the various concentrations of the broth components; altering the OD; incubations times; though some modifications helped none had reproducible effects. However, preliminary tests were preformed on the other processes described. The agarose gel electrophoresis, the southern blotting technique are standard method for DNA amplification experiments. Both the Geneclean and BigDye reaction kits were tested on a similar project and had proven to be successful. Sequence analysis would have been preformed by a professional service so problems related to that process would not be a concern. I fully believe had the vectorization worked the project would have been successful, the anticipated SSRs in the Kachina Daisy genome would have been collected, and the necessary information to help conservationist would have been divulged.