We isolated and characterized 17 tetranucleotide microsatellite loci in the American alligator, Alligator mississippiensis. Loci were screened across 27 individuals from one population and shown to be polymorphic with the number of alleles per locus ranging from 2 to 12. Polymorphic information content ranged from 0.2 to 0.85, and observed heterozygosity ranged from 0.185 to 0.889. One locus showed significant deviation from Hardy–Weinberg equilibrium, and one pair of loci showed evidence of linkage.

Mice of the genus Peromyscus, including several endangered subspecies, occur throughout North America and have been important models for conservation research. We describe 526 primer pairs that amplify microsatellite DNA loci for Peromyscus maniculatus bairdii, 467 of which also amplify in Peromyscus polionotus subgriseus. For 12 of these loci, we report diversity data from a natural population. These markers will be an important resource for future genomic studies of Peromyscus evolution and mammalian conservation.

Ecological studies using microsatellite data often require the selection of an optimal marker set for use in parentage and relatedness inference. Commonly, this requires a candidate pool of microsatellite markers from which several are selected to ensure data are acquired efficiently and accurately. We developed 10 microsatellite loci for use with Northern Bobwhite (Colinus virginianus) and tested these loci using individuals collected from two distinct populations in GA and VA. Our new markers yielded seven alleles/locus (range: 2–16) in the Georgia population and six alleles/locus (range: 2–13) in the Virginia population. Exclusionary power of all markers in each population with both parents unknown was >0.98. These microsatellite loci should be combined with previously developed markers to select an optimal set for use in subsequent analyses of parentage and relatedness.

We have developed eight high-quality microsatellite DNA loci for the saltmarsh sharp-tailed sparrow and one additional locus with evidence of null alleles. In a sample of 250-350 individuals, the average number of alleles per locus was 14.7 and average observed heterozygosity was 0.80. These loci were tested in three additional species of emberizid sparrows, indicating that more than half of the loci could be useful in other sparrows.

Cordylophora caspia, a colonial hydrozoan native to the Ponto-Caspian region, has become a common invader of both fresh and brackish water ecosystems of North America and Europe. We describe 11 polymorphic microsatellite loci for this species. Preliminary analyses indicate that population substructure may contribute to departures from Hardy-Weinberg equilibrium. In addition, new loci failed to consistently amplify Cordylophora samples known to be genetically distant from those utilized in this study, indicating the presence of cryptic diversity within the taxon.

ABSTRACT Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori -specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

We isolated 11 polymorphic microsatellite markers from Acacia mellifera, a savannah woodland tree in sub-Saharan Africa and southern Arabia. The loci were screened for polymorphism using 48 Kenyan individuals. Allelic diversity ranged from three to 19 per locus and the polymorphic information content varied from 0.287 to 0.893. These loci will be useful in studies of genetic structure, gene flow and breeding systems.

A series of techniques are presented to construct genomic DNA libraries highly enriched for microsatellite DNA loci. The individual techniques used here derive from several published protocols but have been optimized and tested in our research laboratories as well as in classroom settings at the University of South Carolina and University of Georgia, with students achieving nearly 100% success. Reducing the number of manipulations involved has been a key to success, decreasing both the failure rate and the time necessary to isolate loci of interest. In our lab during the past 3 years alone, these protocols have been successfully used to isolate microsatellite DNA loci from at least 55 species representing three kingdoms. These protocols have made it possible to reduce the time to identify candidate loci for primer development from most eukaryotic species to as little as 1 week.

The lack of polymorphic genetic markers suitable for genotyping sperm, eggs, and all life stages of the important agricultural pest, Anastrepha suspensa , have prevented detailed genetic studies of its breeding system, reproductive dynamics, and population dynamics. We describe polymerase chain reaction (PCR) primers and reaction conditions for amplifying nine polymorphic microsatellite DNA loci isolated from this species. The PCR primers were tested on four to five individuals each collected from five geographically distant locations in Florida. Heterozygosity values and the number of alleles per locus varied from 0.11 to 0.89, and from two to 12, respectively.

Dollar sunfish (Lepomis marginatus) at the Savannah River Site were sampled from two radiocesium contaminated ponds with a history of thermal elevation and from reference ponds with no history of radioactive or thermal contamination. Fluorescent sequencing of a portion of the mitochondrial control region showed no genetic variation within or between populations. Centrarchidae microsatellite loci Lma 120 ( 6 alleles, N=124 from 5 populations), Lma 20 (10 alleles, N=37 from 3 populations) and Rb 7 (14 alleles. N=l 7 from 2 populations) were variable for dollar sunfish. An additional 9 microsatellite primer pairs were designed for dollar sunfish. Testing of the designed primers on ~20 individuals from 5 populations ~howed these loci have a high number of alleles ranging from 4 to 14 (mean= 8.6) and observed heterozygosities ranged from 0.500 to 0.857 (mean heterozygosity = 0.6337). Age analysis showed most individuals were 1-3 years of age. Only 14 individuals from two contaminated sites, Pond C and the West Arm of PAR, had levels of radiocesium in their muscle tissue above the detection limit. Tissue samples need to be re-measured using larger quantities of tissue for more accurate results in order to correlate radiocesium concentration to fish age. Overall, no comparison of genetic variation could be made for contaminated and uncontaminated ponds using mitochondrial DNA. However, new microsatellite primers for dollar sunfish have been designed for future analyses, and preliminary results indicate that they will be adequate for examining genetic variation for this species. I respectfully dedicate this thesis to my loving parents James and Janet Schable.