Keith R. Merrill and Dr. Craig E. Coleman, BYU Plant and Wildlife Sciences
Introduction
Bromus tectorum (cheatgrass, or downy brome) is an exotic annual weed (recently?) introduced to the United States (ca. 1890) . Since its introduction, B. tectorum has been extremely effective at invading the Intermountain West, displacing native vegetation and causing extensive loss of shrub and rangeland habitats. Because B. tectorum is almost entirely self-pollinating, successful individuals within a given region can be distinguished by genetic analysis. We are using microsatellite markers for genotyping ecologically diverse populations of B. tectorum. Our goal is to better understand the correlation between B tectorum genotypes and the ecological habitats in which they are found, as well as to discover why this species is such a successful invasive. By genotyping various Old World populations (e.g. Greece, Turkey, Cyprus, etc), we seek to locate the origins of the most successful U.S. variants, and thus gain insight into what helps keep B. tectorum in check in these indigenous regions.
Microsatellites
DNA is composed of individual building blocks called nucleotides. There are four different nucleotides, they are: Adenine (A), Cytosine (C), Guanine (G), and Thiamine (T). Microsatellites are areas within the DNA of a species that have short sequence repeats (SSRs) of these nucleotides; e.g. Cytosine and Thiamine repeated numerous times (CTCTCTCTCT). The number of times a sequence is repeated alters the length of that sequence within an individual. Because the length can differ between two different individuals in a species, it is possible to distinguish between two related or unrelated individuals by looking at the length differences at these repeated regions.
Methods and Results
We have been using four microsatellite markers to examine genetically how similar or different populations of B. tectorum are to each other. To date, we have nearly completed a data set containing information on 72 populations (20 samples per population) from throughout the Intermountain West (i.e. the region of the United States between the Rocky Mountains on the east and the Cascade and Sierra Nevada Mountain Ranges on the west). We have also included a few Old World populations from Greece, Turkey, Austria, and Cyprus. Preliminary analysis agrees with previously published results indicating a stronger relationship between populations located in similar habitats than between populations that are only geographically closer to each other. This also supports the prevailing hypothesis that B. tectorum is so successful, at least in part, due to its pre-adaptation to certain habitats and ability to spread rapidly.
Complications
The first hold-up with this project was time. In order to run the microsatellite analyses, DNA had to be extracted from fresh leaf tissue collected from each individual. When I began the project, I was using a proprietary protocol for extractions by which a single person could extract DNA from a maximum of 20 individuals in four hours. This totals just less than 300 hours required to perform the necessary extractions if everything went perfectly without complications. As the project progressed, more samples were added into the study and it quickly became apparent that I needed a faster way to perform the extractions. With the help of Dr. Coleman, I was able to locate a mass-extraction protocol that would allow me to perform 192 extractions in five hours (over 700% increase in efficiency); however, it required a specialized piece of equipment that we did not have and cost over $9,000 retail. Amazingly, I was able to find a used one on E-bay for $1,500. It was a bit beaten, and missing a few parts, but after ordering the necessary parts and working on it for a few hours, everything was working properly. This has been of huge benefit not only to my project but also to other projects in the lab, which have been utilizing the new protocol.
Recently, I discovered that the microsatellite markers we have been using are not nearly as robust as originally assumed. While trying to clear up some ambiguity between some of the different lengths, I decided to directly sequence a few of the troublesome microsatellites. Through sequencing of same-length microsatellites, I have demonstrated that there are multiple repeats within the regions of our microsatellites. This means that a single length may be obtained in more than one way and complicates the analysis I have completed thus far, indicating that genetic diversity of B. tectorum has been underestimated.
Future Research
The scope of this project continues to increase as we add new populations. Most recently we have added recent collections from Old World regions. We are also working on finalizing the data that has already been produced. The problems with our microsatellites have also evoked conversations about the need to develop new, more robust markers for use with B. tectorum. Thus, we are currently planning how to develop single nucleotide polymorphism (SNP) markers. These are more robust and eliminate most of the problems inherent in microsatellite markers. Another long-range goal with B. tectorum is to sequence the genome in order to facilitate cross-species comparison with well-characterized grass species such as rice and maize.
Publication
I will be presenting the results of this study via poster presentation at the Plant and Animal Genome (PAG) XVII Conference in San Diego, CA on January 10-14, 2009. The results will also be submitted for publication to an as yet undecided journal.
Acknowledgements
I would like to thank my mentor, Dr. Craig E. Coleman for his help and direction. I would also like to acknowledge Dr. Susan E. Meyer of the USDA Shrub Sciences Laboratory in Provo, UT and Dr. Mikel R. Stevens of the BYU Plant and Wildlife Sciences Dept for their help and direction. Numerous undergraduates, whose help and labor has been invaluable, have also aided me in this project.