Mitochondrial DNA and Microsatellite Genetic Variation of Dollar Sunfish (Lepomis marginatus)

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=17 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 showed 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

Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water▿

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

Microsatellite loci for the invasive colonial hydrozoan \u3ci\u3eCordylophora caspia\u3c/i\u3e

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

Five hundred microsatellite loci for Peromyscus

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.Organismic and Evolutionary Biolog