Usefulness of molecular markers for detecting population bottlenecks via monitoring genetic change

It is important to detect population bottlenecks in threatened and managed species because bottlenecks can increase the risk of population extinction. Early detection is critical and can be facilitated by statistically powerful monitoring programs for detecting bottleneck-induced genetic change. We used Monte Carlo computer simulations to evaluate the power of the following tests for detecting genetic changes caused by a severe reduction in a population's effective size (Ne): a test for loss of heterozygosity, two tests for loss of alleles, two tests for change in the distribution of allele frequencies, and a test for small Ne based on variance in allele frequencies (the 'variance test'). The variance test was most powerful; it provided an 85% probability of detecting a bottleneck of size Ne = 10 when monitoring five microsatellite loci and sampling 30 individuals both before and one generation after the bottleneck. The variance test was almost 10-times more powerful than a commonly used test for loss of heterozygosity, and it allowed for detection of bottlenecks before 5% of a population's heterozygosity had been lost. The second most powerful tests were generally the tests for loss of alleles. However, these tests had reduced power for detecting genetic bottlenecks caused by skewed sex ratios. We provide guidelines for the number of loci and individuals needed to achieve high-power tests when monitoring via the variance test. We also illustrate how the variance test performs when monitoring loci that have widely different allele frequency distributions as observed in five wild populations of mountain sheep (Ovis canadensis).     

Population substructure and isolation by distance in three continental regions

Isolation by distance and divergence from a shared population history are two sources of population substructure. Isolation by distance erases population history as populations approach migration-drift equilibrium, while diverging populations descended from a single ancestral population will accumulate genetic differences with time. Here I investigate how much of the worldwide genetic diversity from Jorde et al.'s ([1997] Proc. Natl. Acad. Sci. USA 94:3100-3103) 60 tetranucleotide short tandem repeat (STR) data can be explained by isolation by distance. I use Slatkin's measure of population substructure, R(ST), principal components analyses, and Mantel tests to investigate the pattern of genetic diversity at both the intercontinental and intracontinental levels. Geographic distance accounts for almost 60% of world-wide interpopulation genetic relationships. Within continents, the correlations are less, although not significantly so because of wide confidence intervals. These results suggest that populations have not reached migration-drift equilibrium and that there is information in STR data to reconstruct population history. The level of population substructure worldwide is consistent with previous observations, but at the intracontinental level substructure is less. When one examines diversity against distance from the centroid, one sees excess heterozygosity in Africa, a pattern also noted by Stoneking et al. ([1998] Genome Research 7:1061-1071). A larger effective population size in Africa could explain the excess diversity. Greater gene flow in Africa is an unlikely explanation because the African R(ST) value is slightly larger than the Asian and European R(ST)s, pointing to less gene flow and greater substructure among African populations. Furthermore, there are differences in patterns between heterozygosity and allele size variance. Heterozygosity has a higher correlation with distance from the centroid than does allele size variance, and this may reflect demographic history. Kimmel et al. ([1998] Genetics 148:1921-1930) have shown that after a population expansion heterozygosity returns to equilibrium more quickly than does allele size variance. The contrasting patterns between heterozygosity and allele size variance may reflect different times after an expansion. However, simulations and further work need to be done to more thoroughly investigate the possibility that these data reflect population expansions.     

Periodontal considerations in an overdenture population

Three variable microsatellite loci have been isolated from the American lobster, Homarus americanus. In a population sample from the Gulf of Maine, the effective numbers of alleles (Ne) for the two most variable loci were 16.33 and 13.19, respectively. Reduced variability at all three loci was seen in the European lobster, H. gammarus, for which the maximum Ne was 4.00. The reduction in variability in H. gammarus is consistent with a bottleneck event. Inheritance analysis using H. americanus demonstrated segregation of codominant alleles and the absence of linkage. Null alleles were observed at two loci in inheritance studies. This study demonstrates that microsatellite loci should be useful in studying the population structure of clawed lobsters.     

Recurrence of major depressive disorder in hospitalized children and adolescents

Global studies of within-group genetic variation have revealed a tendency for some traits, but not all, to show higher heterozygosity in sub-Saharan African populations. Although excess African diversity has been interpreted as reflecting a greater "age" of sub-Saharan African populations, more recent research has shown that this excess is more likely a consequence of a larger African long-term effective population size. The observation that certain traits, particularly classic genetic markers and RFLPs, do not show this pattern has been interpreted as ascertainment bias. Here, I examine another possible factor: that excess African heterozygosity is in part a function of mutation rate. Simple equilibrium and nonequilibrium models of absolute excess heterozygosity are examined. The results indicate that there is little excess African heterozygosity for traits with low mutation rates and greater excess heterozygosity for traits with moderate to high aggregate mutation rates. Observed data are consistent with these models. Also, depending on population size and time depth, traits with high levels of mutation might show less excess heterozygosity than those with moderate to high mutation rates. Another measure of diversity, mean sequence divergence, shows an increase in excess diversity for traits with high mutation rates.     

Nucleotide sequence diversity at the alcohol dehydrogenase 1 locus in wild barley (Hordeum vulgare ssp. spontaneum): an evaluation of the background selection hypothesis

The background selection hypothesis predicts a reduction in nucleotide site diversity and an excess of rare variants, owing to linkage associations with deleterious alleles. This effect is expected to be amplified in species that are predominantly self-fertilizing. To examine the predictions of the background selection hypothesis in self-fertilizing species, we sequenced 1,362 bp of adh1, a gene for alcohol dehydrogenase (Adh; alcohol:NAD+ oxidoreductase, EC 1.1.1.1), in a sample of 45 accessions of wild barley, Hordeum vulgare ssp. spontaneum, drawn from throughout the species range. The region sequenced included 786 bp of exon sequence (part of exon 4, all of exons 5-9, and part of exon 10) and 576 bp of intron sequence (all of introns 4-9). There were 19 sites polymorphic for nucleotide substitutions, 8 in introns, and 11 in exons. Of the 11 nucleotide substitutions in codons, 4 were synonymous and 7 were nonsynonymous, occurring uniquely in the sample. There was no evidence of recombination in the region studied, and the estimated effective population size (Ne) based on synonymous sites was approximately 1.8-4.2 x 10(5). Several tests reveal that the pattern of nonsynonymous substitutions departs significantly from neutral expectations. However, the data do not appear to be consistent with recovery from a population bottleneck, recent population expansion, selective sweep, or strong positive selection. Though several features of the data are consistent with background selection, the distributions of polymorphic synonymous and intron sites are not perturbed toward a significant excess of rare alleles as would be predicted by background selection.     

The number of self-incompatibility alleles in a finite, subdivided population

The actual and effective number of gametophytic self-incompatibility alleles maintained at mutation-drift-selection equilibrium in a finite population subdivided as in the island model is investigated by stochastic simulations. The existing theory founded by Wright predicts that for a given population size the number of alleles maintained increases monotonically with decreasing migration as is the case for neutral alleles. The simulation results here show that this is not true. At migration rates above Nm = 0.01-0.1, the actual and effective number of alleles is lower than for an undivided population with the same number of individuals, and, contrary to Wright's theoretical expectation, the number of alleles is not much higher than for an undivided population unless Nm < 0.001. The same pattern is observed in a model where the alleles display symmetrical overdominant selection. This broadens the applicability of the results to include proposed models for the major histocompatibility (MHC) loci. For a subdivided population over a large range of migration rates, it appears that the number of self-incompatibility alleles (or MHC-alleles) observed can provide a rough estimate of the total number of individuals in the population but it underestimates the neutral effective size of the subdivided population.     

The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in Leishmania mexicana promastigotes

Microsatellites can be highly unstable and show a high level of polymorphism between individuals. Here we present the analysis of the CAG trinucleotide repeat polymorphism at the SBMA locus in 57 phenotypically normal individuals rigorously assigned to the Spanish Basque population. Results are compared with 100 Spanish non-Basque individuals who were already analyzed by us (175 alleles). This is the first study undertaken in these populations for this marker. In addition, we compared our results with those published for other populations. Relative allele frequencies showed differences between the samples and no unimodal distribution. The expected heterozygosity in the Basque sample was slightly lower than in the non-Basque sample. Conformity with Hardy-Weinberg equilibrium was verified by three tests. When compared with published data, the predominant alleles appear to be the same in the various populations. There are more differences between Basques and other Caucasoid samples than between non-Basques and Caucasoid samples. Population relationships were also examined by dendrograms based on genetic distances. The results obtained showed some peculiarities in the Basque population. The high degree of similarity with other dendrograms based on different markers and the efficiency of this STR marker in differentiating closely related populations, support the potential usefulness of microsatellites as tools for human population studies.     

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.     

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA's D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.     

Isolated limb infusion with cytotoxic agents: a simple alternative to isolated limb perfusion

We use population genetics theory and computer simulations to demonstrate that population bottlenecks cause a characteristic mode-shift distortion in the distribution of allele frequencies at selectively neutral loci. Bottlenecks cause alleles at low frequency (< 0.1) to become less abundant than alleles in one or more intermediate allele frequency class (e.g., 0.1-0.2). This distortion is transient and likely to be detectable for only a few dozen generations. Consequently only recent bottlenecks are likely to be detected by tests for distortions in distributions of allele frequencies. We illustrate and evaluate a qualitative graphical method for detecting a bottleneck-induced distortion of allele frequency distributions. The simple novel method requires no information on historical population sizes or levels of genetic variation; it requires only samples of 5 to 20 polymorphic loci and approximately 30 individuals. The graphical method often differentiates between empirical datasets from bottlenecked and nonbottlenecked natural populations. Computer simulations show that the graphical method is likely (P > .80) to detect an allele frequency distortion after a bottleneck of < or = 20 breeding individuals when 8 to 10 polymorphic microsatellite loci are analyzed.     

Genic homozygosity in an ancient reptile (Alligator mississippiensis)

Proteins assayed electrophoretically showed variation at only three of 49 presumed genetic loci in alligators from southwestern Louisiana. Average heterozygosity per individual was 0.021+/-0.012; proportion of polymorphic loci was 0.06. Data on the history, structure, and ecology of this alligator population are consistent with natural selection as the primary factor accounting for this low genetic variability. However, neither a historic population bottleneck nor some genetic mechanism limiting variability can be dismissed as a possible factor.     

A model of socioeconomic analysis: its construction and results

Genetic structure of 20 of the 37 linguistic groups in the Eastern Highlands of New Guinea including the kuru region is analyzed using information on blood groups and serum protein polymorphisms. The average individual is heterozygous at 28.6% of loci and the average number of alleles per locus is 1.234. Coefficients of kinship for linguistic groups range from 0.005 for the sweet potato cultivating North Fore to 0.075 for the isolated Pawaians whose dietary staple is sago and who depend more on hunting and gathering. As one selects linguistic groups with smaller and smaller population size and increasing isolation, one finds that kinship coefficients rise as much as tenfold, but there is no concomitant loss of heterozygosity or trend toward fixation of alleles. Genetic relationships established by genetic distance trees and by principal components analysis are comparable and are consistent with other anthropological observations.     

Serous cystic neoplasm involving the pancreas and liver: an unusual clinical entity

Allele frequencies for four short tandem repeat loci were determined in a population sample from Catalonia (NE Spain). After denaturing PAGE electrophoresis, 8 alleles were identified for D3S1358 (n=201), 10 alleles for D8S1179 (n=198), 13 alleles for D18S51 (n=197) and 11 alleles for D19S253 (n=201). No deviation from Hardy-Weinberg equilibrium was found. Complete and relative uniformity in Caucasoid populations has been observed for D18S51 and D8S1179 respectively. Pronounced differences were found between different ethnic groups for both systems. Catalonia and Portugal do not differ for D3S1358 locus. Multiplex PCR amplifications of three loci (D3S1358, D18S51 and D19S253) without overlapping fragment size ranges could be interesting for monochrome automated laser fluorescence devices.     

Analysis of allele distribution for six short tandem repeat loci in the French Canadian population of Québec

Short tandem repeat (STR) loci represent a rich source of highly polymorphic markers in the human genome which are useful for the purposes of forensic identification and determination of biological relatedness of individuals. Here, as a part of an ongoing extensive study, we report the analysis of a multilocus genotype survey of 642 to 870 chromosomes in the French Canadian Caucasian population of Québec at six STR loci. The loci HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13A01, HUMFESFPS, and HUMvWA were typed using two multiplex polymerase chain reactions (PCR). Amplified DNA samples were subsequently analyzed by polyacrylamide gel electrophoresis followed by silver staining. The heterozygote frequencies of the loci range from 0.614 to 0.820 (0.661 to 0.818 expected) and the number of alleles from 7 to 12 per locus. Although statistically significant deviation from Hardy-Weinberg expectations of genotype frequencies was noted at some loci by one or more tests, in general, the genotype frequencies are well estimated from the product of allele frequencies at all loci. The most frequent six-locus genotype is expected to occur in the French Canadian population with a frequency of 3.50 by 10(-5) and together, these six loci have an average probability of discrimination of 0.9999985. The study presented here indicates that these six STR loci are informative genetic markers for identity testing purposes in the French Canadian Caucasian population of Québec.     

Scarlet fever in 1996]

A sample population of Rio de Janeiro, Brazil, was tested for D6S132, D7S467, and D17S26 VNTR loci to determine the fixed-bin frequencies of alleles. RFLP analysis was obtained by HaeIII-digested genomic DNA. The three VNTR loci meet Hardy-Weinberg expectations, and there is no evidence for association of alleles between the VNTR loci. The frequency data can be used in forensic analysis, in paternity tests and to estimate the frequency of a DNA profile in the general Brazilian population.     

Combination of irinotecan (CPT11) and 5-fluorouracil with an analysis of cellular determinants of drug activity

Historical population collapses caused by rinderpest epidemics are hypothesized to have resulted in notable genetic losses in populations of the African buffalo. Polymorphism in the major histocompatibity complex (MHC) DRB3 gene was probed by means of restriction analysis of the sequence encoding the peptide-binding region. Nucleotide substitution patterns agreed with a positive selection acting on this fitness-relevant locus. Buffalo populations from four National Parks, situated in eastern and southern Africa, each revealed a surprisingly high allelic diversity. Current high levels of heterozygosity may be reconciled with historical bottlenecks by assuming that local extinctions were followed by fast recolonization, in accordance with the high dispersive capabilities of buffalo. The specific amplification of DRB3 alleles also enabled the assignment of individual genotypes. For each population sample a deficiency in the expected number of heterozygous animals was found. As overdominant selection on the MHC is predicted to yield an excess of heterozygous individuals, this may not be a locus-specific effect. Several other explanations are discussed, of which increased homozygosity caused by nonrandom mating of buffalo in populations seems the most probable.     

Swiss population data and forensic efficiency values on 3 tetrameric short tandem repeat loci-HUMTH01, TPOX, and CSF1PO-derived using a STR multiplex system

Allele and genotype frequencies for 3 tetrameric short tandem repeat loci were determined in a Swiss population sample (n = 100) using the GenePrint STR Multiplex System, electrophoresis of the PCR products in DNA sequencing gels and subsequent detection of allelic fragments by silver staining. The loci are HUMTH01, TPOX, and CSF1PO. The observed heterozygosities are 83.0%, 60.0%, and 72.0%, respectively. The discrimination power determined for the individual loci is 0.914, 0.780, and 0.860, respectively, and the combined discrimination power for the triplex is 0.997. All loci meet Hardy-Weinberg expectations and after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence. Moreover, independence of alleles at these STR loci with other PCR-based loci derived from the same Swiss population sample, previously reported, were considered. These loci were DQA1, LDLR, GYPA, HBGG, D7S8, GC and D1S80. Again, after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence among alleles at the 10 different PCR-based loci. Thus, the allelic frequency data can be used in human identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Swiss population.     

Effect of a founder event on variation in the genetic sex-determining system of the fire ant Solenopsis invicta

Effects of a recent founder event on genetic diversity in wild populations of the fire ant Solenopsis invicta were studied, with particular attention given to the genetic sex-determining system. Diploid males are far more common relative to haploid males in introduced populations than in native populations of fire ants, and queens that produce diploid males account for a significantly larger proportion of the mated queens in introduced than in native populations. Differences between native and introduced populations in attributes of the mating systems (i.e., queen mating frequency or level of inbreeding) can be excluded as factors contributing to these different levels of diploid male production. Thus, we conclude that diploid males have increased in frequency in introduced populations because of a loss of allelic diversity at the sex-determining locus (loci). This loss of sex alleles has generated a substantial increase in the estimated segregational genetic load associated with production of sterile diploid males in introduced populations over the load in native populations. The loss of allelic diversity in the sex-determining system in introduced S. invicta is paralleled by a loss of electrophoretically detectable rare alleles at protein-encoding loci. Such concordance between these different types of markers is predicted because each of the many sex alleles present in the native populations is expected to be rare. Estimates of expected heterozygosity (Hexp) based on 76 electrophoretic loci do not differ significantly between the native and introduced fire ant populations, illustrating the lack of sensitivity of this measure for detecting many types of bottlenecks.     

Sequence-tagged-site (STS) markers of arbitrary genes: development, characterization and analysis of linkage in black spruce

Sequence-tagged-site (STS) markers of arbitrary genes were investigated in black spruce [Picea mariana (Mill.) B.S.P.]. Thirty-nine pairs of PCR primers were used to screen diverse panels of haploid and diploid DNAs for variation that could be detected by standard agarose gel electrophoresis without further manipulation of amplification products. Codominant length polymorphisms were revealed at 15 loci. Three of these loci also had null amplification alleles as did 3 other loci that had no apparent product-length variation. Dominant length polymorphisms were observed at 2 other loci. Alleles of codominant markers differed in size by as little as 1 bp to as much as an estimated 175 bp with nearly all insertions/deletions found in noncoding regions. Polymorphisms at 3 loci involved large (33 bp to at least 114 bp) direct repeats and similar repeats were found in 7 of 51 cDNAs sequenced. Allelic segregation was in accordance with Mendelian inheritance and linkage was detected for 5 of 63 pairwise combinations of loci tested. Codominant STS markers of 12 loci revealed an average heterozygosity of 0.26 and an average of 2.8 alleles in a range-wide sample of 22 trees.