Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method.

Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition.

A population genetic comparison of argali sheep (Ovis ammon) in Mongolia using the ND5 gene of mitochondrial DNA; implications for conservation.

We sequenced 556 bp of the mitochondrial ND5 gene to infer aspects of population structure and to test subspecific designations of argali sheep (Ovis ammon) in Mongolia. Analysis of molecular variance (AMOVA) revealed greater variation within than among putative subspecies and populations, suggesting high levels female-mediated gene flow. Compared with bighorn sheep (O. canadensis) in North America, substantially less differentiation in mitochondrial DNA was found among argali populations over 1200 km than was found among bighorn populations over 250 km. This result is consistent with differences in argali and bighorn life history traits. Argali run for long distances across open terrain in the presence of a threat rather than running up into steep escape terrain like bighorn sheep do. Our results suggest recognizing only one Evolutionary Significant Unit (subspecies) of argali in Mongolia, but they may support recognizing two Management Units, because two regions do exhibit slightly different haplotype frequencies at the ND5 gene of mtDNA.

Phylogeny and host specificity of Psoroptic mange mites (Acarina: Psoroptidae) as indicated by ITS sequence data.

We used molecular phylogenetic techniques to study the systematic relationships and host specificity of Psoroptes mange mites, which are pests of numerous domestic and wild ungulates. Phylogenetic analysis of DNA sequence data from the internal transcribed spacer region 1 (ITS1) of nuclear ribosomal DNA indicated that populations of Psoroptes are not host specific. Furthermore, the currently used taxonomy of Psoroptes is not concordant with the phylogeny derived from ITS1. During the course of the study, we discovered apparent paralogous ITS sequences within individual mites as a result of varying polymerase chain reaction reaction conditions. This finding concords with other studies of ITS and suggests a cautious approach when interpreting data from ITS sequences. Host DNA contamination was also found to be a significant problem in data collection, and we report on the development of methods to overcome the problems of contamination in parasitic mites.

Population subdivision among desert bighorn sheep (Ovis canadensis) ewes revealed by mitochondrial DNA analysis.

We used behavioural observations and mitochondrial DNA (mtDNA) sequence analysis to examine demographic and genetic structure within and among home-range groups of desert bighorn sheep (Ovis canadensis) ewes in the Peninsular Ranges of southern California, USA. We identified substantial genetic variation in the first 515 bp of the mtDNA control region and determined that seven haplotypes were distributed in a nonrandom fashion among these ewe subpopulations. Although a significant (P < 0.01) amount of mtDNA variation (33%) was partitioned among home-range groups, we did not find strong evidence for matrilineal substructuring within these groups. Based on analyses of molecular variance, and comparisons of behavioural associations and distances between centres of activity, we concluded that within a given home-range group, bighorn sheep ewes generally associate with other ewes based on their availability rather than their matrilineal relationships. Our results also supported the conclusion that multiple ewe subpopulations exist within the Peninsular Ranges, and that these subpopulations are the most basic demographic and genetic units.

Resistance mutations in protease and reverse transcriptase genes of human immunodeficiency virus type 1 isolates from patients with combination antiretroviral therapy failure.

High-density oligonucleotide arrays were used to determine the sequence of the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 isolates from 35 patients in whom combination therapy that included a protease inhibitor had failed. Isolates had a median of three PR mutations (range, none to six). Three isolates had no known resistance mutations in PR. Twelve isolates (34%) had two or fewer resistance mutations in PR. The most commonly observed PR mutations were L10I, V82A/T/F, and L90M. No mutations were observed at codons 30 or 48. Mutations at RT codons 215 and 184 were observed in the majority of isolates. These data suggest that therapy can fail in some patients with relatively few PR resistance mutations. Clinical failure in the absence of resistance mutations implies inadequate drug exposure due to pharmacologic factors or suboptimal patient adherence to drug therapy.

Genetic variation of major histocompatibility complex and microsatellite loci: a comparison in bighorn sheep.

Examining and comparing genetic variation for major histocompatibility complex (MHC) and micro-satellite (MS) loci in the same individuals provides an opportunity to understand the forces influencing genetic variation. We examined five MHC and three MS loci in 235 bighorn sheep (Ovis canadensis) from 14 populations and found that both types of loci were highly variable and were in Hardy-Weinberg proportions. Mean FST values for both markers were very similar and MHC and MS genetic variability was predominantly distributed within rather than among populations. However, analyses of genetic distances and tree topologies revealed different spatial patterns of variation for the two types of loci. Collectively, these results indicated that neutral forces substantially influenced MS and MHC variation, and they provided limited evidence for selection acting on the MHC.

Mitochondrial DNA variation, population structure, and evolution of mountain sheep in the south-western United States and Mexico.

In the south-western United States and Mexico, mountain sheep were found to have low levels of mtDNA sequence divergence among haplotypes and low overall nucleotide diversity. Potential causes for this low diversity are discussed. Significant differences in mtDNA haplotype distributions over short distances and high values of NST on a local scale appear to be a result of the tendency of female mountain sheep to disperse less frequently and over shorter distances than males. A lack of concordance between mtDNA haplotype distributions and assumed subspecies boundaries suggest that some taxonomic labels need to be revised.