Kinetic analyses as a critical parameter in defining the side population (SP) phenotype

The side population (SP) phenotype has been reported as a method to identify hematopoietic stem cells in the bone marrow based upon differential staining with the fluorescent dye, Hoechst 33342. This technique has drawn great interest in the stem cell community, as it may provide a simple approach to the enrichment of progenitor cells from a variety of normal and malignant tissues. The frequency of these cells and their performance in functional assays has varied considerably within the literature. To investigate mechanisms that may contribute to the SP phenotype, we measured the fluorescence emission of Hoechst-stained bone marrow cells as a function of both time and dye concentration using a custom flow cytometer and data acquisition software. These measurements demonstrate that all nucleated cells within the bone marrow undergo an identical staining pattern at varying rates, even under conditions previously reported to abrogate the SP. Therefore, the SP phenotype is not unique to stem cells, but rather represents a transient feature of marrow cells exposed to Hoechst 33342 for varying amounts of time. We propose that heterogeneity of SP-defined populations may be a consequence of the rate at which differing cell populations accumulate Hoechst 33342. Further, we suggest that dye uptake kinetics will likely be an important factor for optimal use of Hoechst 33342 in isolating stem cells.     

Adaptive population differentiation in phenology across a latitudinal gradient in European aspen (Populus tremula, L.): a comparison of neutral markers, candidate genes and phenotypic traits

A correct timing of growth cessation and dormancy induction represents a critical ecological and evolutionary trade-off between survival and growth in most forest trees (Rehfeldt et al. 1999; Horvath et al. 2003; Howe et al. 2003). We have studied the deciduous tree European Aspen (Populus tremula) across a latitudinal gradient and compared genetic differentiation in phenology traits with molecular markers. Trees from 12 different areas covering 10 latitudinal degrees were cloned and planted in two common gardens. Several phenology traits showed strong genetic differentiation and clinal variation across the latitudinal gradient, with Q(ST) values generally exceeding 0.5. This is in stark contrast to genetic differentiation at several classes of genetic markers (18 neutral SSRs, 7 SSRs located close to phenology candidate genes and 50 SNPs from five phenology candidate genes) that all showed F(ST) values around 0.015. We thus find strong evidence for adaptive divergence in phenology traits across the latitudinal gradient. However, the strong population structure seen at the quantitative traits is not reflected in underlying candidate genes. This result fit theoretical expectations that suggest that genetic differentiation at candidate loci is better described by F(ST) at neutral loci rather than by Q(ST) at the quantitative traits themselves.     

Effects of population size and mutation rate on the evolution of mutational robustness

It is often assumed that the efficiency of selection for mutational robustness would be proportional to mutation rate and population size, thus being inefficient in small populations. However, Krakauer and Plotkin (2002) hypothesized that selection in small populations would favor robustness mechanisms, such as redundancy, that mask the effect of deleterious mutations. In large populations, by contrast, selection is more effective at removing deleterious mutants and fitness would be improved by eliminating mechanisms that mask the effect of deleterious mutations and thus impede their removal. Here, we test whether these predictions are supported in experiments with evolving populations of digital organisms. Digital organisms are self-replicating programs that inhabit a virtual world inside a computer. Like their organic counterparts, digital organisms mutate, compete, evolve, and adapt by natural selection to their environment. In this study, 160 populations evolved at different combinations of mutation rate and population size. After 10(4) generations, we measured the mutational robustness of the most abundant genotype in each population. Mutational robustness tended to increase with mutation rate and to decline with population size, although the dependence with population size was in part mediated by a negative relationship between fitness and robustness. These results are independent of whether genomes were constrained to their original length or allowed to change in size.     

Isolation and characterization of tetranucleotide microsatellite markers in a mouth‐brooding haplochromine cichlid fish (Pseudocrenilabrus multicolor victoriae) from Uganda

Eight tetranucleotide microsatellite loci were isolated from the haplochromine cichlid fish, Pseudocrenilabrus multicolor victoriae, an important model species for studies in respiratory ecology, conservation, and evolution. We surveyed variation at these loci in 23 individuals from western Uganda, finding four to 19 alleles per locus and an average expected heterozygosity of 0.8575. These microsatellite loci will be used to examine gene flow and population structure in Ugandan P. m. victoriae.     

Identification and characterization of eight microsatellite loci in Aster amellus L. (Asteraceae)

Eight polymorphic microsatellites were developed in the perennial herbaceous Aster amellus L. (Asteraceae) and characterized on three populations from France and Switzerland. The number of alleles ranged between four and 30 depending on the locus, and the mean number of effective alleles was 5.8. The average gene diversity equalled 0.744 (range: 0.419–0.957) and the overall differentiation was found significant (θ = 0.092, P < 0.01). Three loci displayed significant heterozygote deficiencies, which might indicate the presence of null alleles. Amplifications were detected on eight loci in Aster alpinus L.     

Development and characterization of nine polymorphic microsatellite markers in the sand goby Pomatoschistus minutus (Gobiidae)

A microsatellite‐enriched genomic library was constructed for the sand goby, Pomatoschistus minutus (Pallas 1770), and nine polymorphic DNA microsatellite markers of high quality were successfully optimized. Characterization of 96 individuals from the Vaccarès lagoon (France) showed moderate to high levels of polymorphism (two to 54 alleles). All the markers conformed to Hardy–Weinberg equilibrium and showed no evidence of null alleles, large allele dropout, stuttering and linkage disequilibrium between pairs of loci. These markers successfully amplify in three closely related species and can be employed to investigate population genetic structure and to clarify paternity in Pomatoschistus species.     

Isolation and characterization of polymorphic microsatellite loci for the study of paternity and population structure in the little penguin Eudyptula minor

We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction‐based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population (mean H_E = 0.613, mean N_A = 7.14). Cross‐amplification using a microsatellite primer developed in Spheniscus demersus (African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins (exclusion probability for seven unlinked loci = 0.993).     

Isolation and characterization of six microsatellites in the Mexican beaded lizard Heloderma horridum

Six polymorphic microsatellite loci were isolated and characterized in the threatened Mexican beaded lizard, Heloderma horridum. The number of alleles per locus ranged from three to 12, with observed heterozygosity estimates ranging from 0.00 to 0.77, and expected heterozygosity estimates ranging from 0.00 to 0.73. These microsatellites will provide a valuable tool for the investigation of the genetic variation and structure of both wild and captive H. horridum populations.     

Microsatellite‐enriched genomic libraries as a source of polymorphic loci for Schistosoma mansoni

Microsatellite markers for Schistosoma mansoni were developed using four genomic microsatellite‐enriched libraries. Microsatellites were observed in 65.4% of all sequences. Primer pairs were designed and tested for 23 loci. Eighteen loci produced amplification products, out of which 11 were polymorphic and were further characterized on 100 individuals of S. mansoni. Two to 19 alleles per locus were detected. The average values of expected and observed heterozygosities among the 11 loci were 0.79 and 0.59, respectively.