Enzyme Variability in Natural Populations of DAPHNIA MAGNA III. Genotypic Frequencies in Intermittent Populations

In temporary habitats populations of the cyclical parthenogen, Daphnia magna, are re-established each year from sexual eggs and reproduce parthenogenetically for two or at most three generations. The genetic effects of this breeding system have been investigated by analyzing allozyme frequencies in nineteen intermittent populations.—Genotypic frequencies at polymorphic loci were ordinarily found to be in good agreement with Hardy-Weinberg proportions and disequilibria between loci were not observed. Although significant changes in gene frequencies were observed both during and between successive cycles, there was no evidence of the marked instability of genotype frequencies characteristic of permanent populations. The recombinational degradation of genotypes at the end of each annual cycle in temporary habitats effectively prevents the genotypic structuring which develops when continued parthenogenesis is possible.     

Enzyme Polymorphism and Cyclic Parthenogenesis in DAPHNIA MAGNA. I. Selection and Clonal Diversity

Genotype frequencies and fecundities were recorded over a period of two years for three polymorphic enzyme loci (Est, Mdh and Got) in a parthenogenetic natural population of Daphnia magna Straus (Crustacea: Cladocera). There was a large excess of heterozygotes at each locus, and some nonrandom association between loci, although 29 different three-locus genotypes were detected. There were small but significant changes in genotype frequencies that did not follow any clear seasonal cycles or overall trends, and the genotypes often differed significantly in fecundity, although the direction of the difference was not constant. These fitness differences were probably not attributable to the specific loci studied.--Models of balancing selection are of two types: segregation-balanced (e.g., heterosis) and competition-balanced (e.g., frequency dependence). Only the latter type can stabilize diversity in a clonal population. The observed selection was not heterotic, but it is not certain that it was stabilizing either. Clonal competition did not lead to victory by a single, fittest clone; genotypic diversity remained high.     

Enzyme Variability in Natural Populations of DAPHNIA MAGNA II. Genotypic Frequencies in Permanent Populations

In permanent habitats populations of the cyclical parthenogen, Daphnia magna, reproduce by continued parthenogenesis and are subject to only sporadic sexual recruitment. The genetic effects of this breeding system have been investigated by analyzing allozyme frequencies in thirteen populations of D. magna.-Genotypic frequencies at polymorphic loci characteristically deviated markedly from Hardy-Weinberg proportions and gametic phase imbalance between loci was frequent. Genotypic frequencies were subject to violent, selectively determined oscillations over short periods of time. These observations suggest that permanent populations of D. magna ordinarily consist of a limited number of highly structured genotypes. The adaptational advantages offered by such structuring may have been a major selective factor favoring the evolution of cyclical parthenogenesis.     

Epistasis of quantitative trait loci under different gene action models

Modeling and detecting nonallelic (epistatic) effects at multiple quantitative trait loci (QTL) often assume that the study population is in zygotic equilibrium (i.e., genotypic frequencies at different loci are products of corresponding single-locus genotypic frequencies). However, zygotic associations can arise from physical linkages between different loci or from many evolutionary and demographic processes even for unlinked loci. We describe a new model that partitions the two-locus genotypic values in a zygotic disequilibrium population into equilibrium and residual portions. The residual portion is of course due to the presence of zygotic associations. The equilibrium portion has eight components including epistatic effects that can be defined under three commonly used equilibrium models, Cockerham's model, F2-metric, and F(infinity)-metric models. We evaluate our model along with these equilibrium models theoretically and empirically. While all the equilibrium models require zygotic equilibrium, Cockerham's model is the most general, allowing for Hardy-Weinberg disequilibrium and arbitrary gene frequencies at individual loci whereas F2-metric and F(infinity)-metric models require gene frequencies of one-half in a Hardy-Weinberg equilibrium population. In an F2 population with two unlinked loci, Cockerham's model is reduced to the F2-metric model and thus both have a desirable property of orthogonality among the genic effects; the genic effects under the F(infinity)-metric model are not orthogonal but they can be easily translated into those under the F2-metric model through a simple relation. Our model is reduced to these equilibrium models in the absence of zygotic associations. The results from our empirical analysis suggest that the residual genetic variance arising from zygotic associations can be substantial and may be an important source of bias in QTL mapping studies.     

Genetic stability in a population of a plant pathogenic fungus over time

Collections of the plant pathogenic fungus Mycosphaerella graminicola were made from the same field of wheat over a 3-year period. The field was planted with small plots containing four varieties of wheat grown in pure stand and in all possible two-, three- and four-way mixtures. In each year, the wheat field was recolonized by a local source of inoculum of unknown origin. Allele frequencies at 10 RFLP loci were compared at two different times within a growing season and over the 3-year period. No significant differences in allele frequencies were found for any of the RFLP loci over any of the time periods. DNA fingerprints were used to identify clones produced by asexual reproduction. Genotypic diversity based on the frequency of each clone was compared for each collection. No significant changes in genotypic diversity were found within a year or between years. Identical genotypes were found in the field at different times within a season, but no clones were conserved between years. No clone existed in a high frequency in any year, suggesting that selection for particular asexual lineages was weak. The founding population each year probably originated from wind-borne ascospores of the teleomorph, which may exist as an indigenous population on alternative hosts, such as Poa annua (annual bluegrass).     

Seasonal and annual genotypic variation and the effect of climate on population genetic structure of the cereal aphid Sitobion avenae in northern France

Changes in the genetic structure and genotypic variation of the aphid Sitobion avenae collected from cereal crops in northern France were examined by analysing variation at five microsatellite loci across several years and seasons. Little regional and temporal differentiation was detected, as shown by very low FST among populations. Repeated genotypes, significant heterozygote deficits, positive FIS values and frequent linkage disequilibria were found in nearly all samples, suggesting an overall pattern of reproductive mode variation in S. avenae populations. In addition, samples from Brittany (Bretagne) showed greater signs of asexual reproduction than those from the north of France, indicating a trend toward increasing sexuality northward. These patterns of reproductive variation in S. avenae are consistent with theoretical models of selection of aphid reproductive modes by climate. Contrasting with little changes in allelic frequencies, genotypic composition varied substantially in time and, to a lesser extent, in space. An important part of changes in genotypic arrays was due to the variation in frequency distribution of common genotypes, i.e. those that were found at several instances in the samples. Genotypic composition was also shown to vary according to climate, as genotypic diversity in spring was significantly correlated with the severity of the previous winter and autumn. We propose that the genetic homogeneity among S. avenae populations shown here across large temporal and spatial scales is the result of two forces: (i) migration conferred by high dispersal capabilities, and (ii) selection over millions of hectares of cereals (mostly wheat) bred from a narrow genetic base.     

Genotypic selection in Daphnia populations consisting of inbred sibships

The genetic basis of fitness reduction associated with inbreeding is still poorly understood. Here we use associations between allozyme genotypes and fitness to investigate the genetic basis of inbreeding depression in experimental outdoor populations of the water flea, Daphnia magna. In Daphnia, a phase of clonal reproduction follows hatching from sexually produced resting eggs, and changes in genotype frequencies during the clonal phase can be used to estimate fitness. Our experiment resembles natural colonization of ponds in that single clones colonize an empty pool, expand asexually and produce sexual offspring by selfing (sisters mate with their clonal brothers). These offspring diapause and form populations consisting of selfed sibships in the following spring. In 12 of 13 experimental populations, genotypes of selfed hatchlings after diapause conformed to Mendelian expectations. During the subsequent ca. 10 asexual generations, however, genotype frequencies changed significantly at 19 of 27 single loci studied within populations, mostly in favour of heterozygotes, with heterozygosity at multiple loci affecting the change in genotype frequency multiplicatively. Because variance in heterozygosity among siblings at a given marker reflects only heterozygosity in the chromosomal region around this marker, our results suggest that selection at fitness-associated loci in the chromosomal regions near the markers were responsible for these changes. The genotype frequency changes were more consistent with selection acting on linked loci than on the allozymes themselves. Taken together, the evidence for abundant selection in the chromosomal regions of the markers and the fact that changes in genotype frequencies became apparent only after several generations of clonal selection, point to a genetic load consisting of many alleles of small or intermediate effects, which is consistent with the strong genetic differentiation and repeated genetic bottlenecks in the metapopulation from which the animals for this study were obtained.     

Temporal dynamics of genotypic diversity reveal strong clonal selection in the aphid Myzus persicae

Parthenogenetic organisms often harbour substantial genotypic diversity. This diversity may be the result of recurrent formations of new clones, or it may be maintained by environmental heterogeneity acting on ecological differences among clones. In aphids, both processes may be important because obligate and cyclical parthenogens can form mixed populations. Using microsatellites, I analysed the temporal dynamics of clonal diversity in such a population of the aphid Myzus persicae over a 1-year period. The frequency distribution of clonal genotypes was very skewed, with many rare and few common clones. The relative frequencies of common clones underwent strong and rapid changes indicative of intense clonal selection. Differences in their host associations suggest that these shifts may partly be caused by changes in the abundance of annual host plants. Other selective factors of potential importance are also discussed. New, sexually produced genotypes made a minor contribution to clonal diversity, consistent with the observed heterozygote excess characteristic of predominantly asexual populations in M. persicae.     

Single nucleotide polymorphisms of estrogen receptor alpha in human renal cell carcinoma

Estrogens and their receptors (ERs) have been shown to play a role in various cancers. We hypothesize that polymorphisms and genotypic changes of the ERalpha gene are involved in renal cell carcinoma. To test this hypothesis, DNA samples from 113 cases of human renal cell cancer were analyzed by sequence-specific polymerase chain reaction to determine the genotypic frequency of six different polymorphic loci on ERalpha gene (codon 10 T-->C, codon 87 G-->C, codon 243 C-->T, codon 325 C-->G, codon 594 G-->A, and intron 1 C-->T). The relative risk of variant genotype was calculated by comparison with 200 healthy controls. The results of this study demonstrate that the distribution of genotypes on codon 10 differs between renal cancer patients and healthy normal controls (p<0.05). The relative risk of the genotype 10C/C was calculated as 2.51. No differences in genotypes were observed at all other loci. We also analyzed DNA from pairs of cancerous and normal tissues from 96 cases of human renal cell cancer to characterize genotypic changes at these loci. Genotypic changes were detected in nine cancer samples on exon 1 (codons 10 and 87) of ERalpha, although none were detected at other regions. The present study demonstrates for the first time that codon 10 polymorphism on exon 1 of ERalpha may be involved in renal cancer risk.     

Direct evidence for biased gene diversity estimates from dominant random amplified polymorphic DNA (RAPD) fingerprints

The relevance of using dominant random amplified polymorphic DNA (RAPD) fingerprints for estimating population differentiation was investigated when typically small population sample sizes were used. Haploid sexual tissues were first used to determine genotypes at RAPD loci for 75 eastern white pines ( Pinus strobus L.) representing five populations. Dominant RAPD fingerprints were then inferred from genotypic data for each individual at each locus, and gene diversity estimates from both sources of data were compared. Genotypic information at RAPD loci indicated little or no differentiation among populations, similar to allozyme loci. However, estimates of population differentiation derived from dominant RAPD fingerprints according to various common methods of analysis were generally inflated, especially when all fragments were considered. Simulations showed that an increase in loci sampling and population sample sizes did not significantly alleviate the biases observed.     

The multiple-locus symmetric fertility model

Selection due to variation in the fecundity among matings of genotypes with respect to many loci each with two alleles is studied. The fitness of a mating depends only on the genotypic distinction between homozygote and heterozygote at each locus in the two individuals, and differences among loci are allowed. This symmetric fertility model is therefore a generalization of the multiple-locus symmetric viability model. The phenomena seen in the two-locus symmetric fertility model generalize—e.g., the possibility of joint stability of equilibria with linkage equilibrium and with linkage disequilibrium, and the existence of different types of totally polymorphic equilibria with the gametic proportions in linkage equilibrium. The central equilibrium with genotypic frequencies in Hardy-Weinberg proportions and gametic frequencies in Robbins proportions exists for all symmetric fertility models. For some symmetric fertility regimes additional equilibria exist with gametic frequencies in linkage equilibrium and with genotypic frequencies in Hardy-Weinberg proportions at all except one locus. These equilibria may exist in the dioecious symmetric viability model, and then they will be locally stable. For free recombination the stable equilibria show linkage equilibrium, but several of these with different numbers of polymorphic loci may be stable simultaneously.     

Testing for linkage disequilibrium in the New Zealand radiata pine breeding population

Linkage analysis is commonly used to find marker-trait associations within the full-sib families of forest tree and other species. Study of marker-trait associations at the population level is termed linkage-disequilibrium (LD) mapping. A female-tester design comprising 200 full-sib families generated by crossing 40 pollen parents with five female parents was used to assess the relationship between the marker-allele frequency classes obtained from parental genotypes at SSR marker loci and the full-sib family performance (average predicted breeding value of two parents) in radiata pine (Pinus radiata D. Don). For alleles (at a marker locus) that showed significant association, the copy number of that allele in the parents was significantly correlated, either positively or negatively, with the full-sib family performance for various economic traits. Regression of parental breeding value on its genotype at marker loci revealed that most of the markers that showed significant association with full-sib family performance were not significantly associated with the parental breeding values. This suggests that over-representation of the female parents in our sample of 200 full-sib families could have biased the process of detecting marker-trait associations. The evidence for the existence of marker-trait LD in the population studied is rather weak and would require further testing. The exact test for genotypic disequilibrium between pairs of linked or unlinked marker loci revealed non-significant LD. Observed genotypic frequencies at several marker loci were significantly different from the expected Hardy-Weinberg equilibrium. The possibilities of utilising marker-trait associations for early selection, among-family selection and selecting parents for the next generation of breeding are also discussed.     

Recombination and genetic differentiation in the mycorrhizal fungus Cenococcum geophilum Fr

Population genetic analyses of the mycorrhizal fungus Cenococcum geophilum were conducted to test for a clonal or recombining population structure. Multilocus genotypes based on polymorphisms in 9 loci, identified in this study by PCR-SSCP techniques, were obtained for two populations. Genotypic variation occurred on a fine scale because unique genotypes were identified at most every transect point, and in some cases occurred even within one soil sample (equivalent to about a 500 mL volume). The largest genet observed occurred over a 30 meter transect space. The two population genetic methods employed to distinguish between clonality and recombination, (1) Index of Association; and (2) "Parsimony Tree Length Permutation Test" (PTLPT), could not reject the null hypothesis of recombination in either population. Wright's Fst, as estimated by theta, was used to examine gene flow between the two populations based on allele frequencies. Two of the nine loci had theta values that were not significantly different from what one would expect for the null hypothesis of panmixia. However, the other seven loci were consistent with reduced gene flow. The theta value for the Fisher combined probability (combining all 9 loci) was significant and indicated that there was genetic differentiation between these two populations.     

The consequences of fluctuating selection for isozyme polymorphisms in Daphnia

Temporal sequences of allele frequencies in natural populations of Daphnia are analyzed to obtain the mean and variance of the selection coefficient for both asexual and sexual phases. In general, the alleles at enzyme loci appear to be quasi-neutral. Although significant variation exists for the estimated selection coefficients, the means are in all cases close to zero. Estimates of the variance of selection intensity are applied to existing models to demonstrate the implications of fluctuating selection for the spatial and temporal distribution of gene frequencies in Daphnia. The empirical and analytical results are shown to provide a possible solution to some previously puzzling aspects of Daphnia population genetic surveys. Neither genetic drift nor diversifying selection are necessary conditions for the local diversification of gene frequencies.     

Allozyme-Associated Heterosis in Drosophila Melanogaster

Two large experiments designed to detect allozyme-associated heterosis for growth rate in Drosophila melanogaster were performed. Heterosis associated with allozyme genotypes may be explained either by functional overdominance at the allozyme loci, or closely linked loci; or by genotypic correlations between allozyme loci and loci at which deleterious recessive alleles segregate. Such genotypic correlations would be favored by consanguineous mating, small effective population size, population mixing and strong natural or artificial selection. D. melanogaster is outbred, has large effective population size and there is little evidence for genotypic disequilibria. Therefore it would be unlikely to show allozyme heterosis due to genotypic correlations. In the first experiment I estimated the genotypic values of 97 replicated genotypes. In the second experiment, 500 individuals were raised in a fluctuating, stressful environment. In neither experiment was there any consistent evidence for allozyme heterosis in size or development rate, fluctuating asymmetry for size or in tendency to deviate from the population mean. In the first experiment, heterosis explained less than 5.6% of the genetic variance in growth characters. In the second, heterosis explained less than 0.1% of the phenotypic variance in growth characters. Outside of the molluscs, species which show allozyme heterosis have population structures or histories which tend to promote genotypic correlations. There is little evidence that functional overdominance is responsible for observations of allozyme-associated heterosis.     

Fitness of allozyme variants in Drosophila pseudoobscura III. Possible factors contributing to the maintenance of polymorphisms in nature

Experiments have been performed to investigate the mechanisms maintaining enzyme polymorphisms in natural populations. We have measured effects on fitness of genotypic variants at three loci, Est-5, Odh, and Mdh-2, in D. pseudoobscura. Significant differences exist among the genotypes in the rate of development from egg-to-adult; there is also indication of differences in larval survival. In a population segregating for allelic variants at all three loci, there is indication that segregation distortion at meiosis or some form of gametic selection might be involved. The relative fitnesses of alternative genotypes are reversed when either different fitness components are considered, or the genotypic frequencies are changed, or the larval density is increased. These fitness reversals may contribute to the maintenance of the polymorphisms, and may account for cyclical oscillations of allozyme frequencies observed in natural populations.Research supported by U.S. Public Health Service Research Fellowship (1F05 TWO 1991-01) to D.M. and by contract AT(04-3)34 with the U.S. Atomic Energy Commission. Adress reprint requests from Europe to D.M.; from elsewhere to F.J.A.     

<Emphasis Type="Italic">ABCB1</Emphasis> single nucleotide polymorphisms in the Brazilian population

The ABCB1/MDR-1 gene, which encodes P-glycoprotein, is highly polymorphic. We analyzed 278 healthy Brazilian individuals through PCR–RFLP to identify ABCB1 variants and determine the genotypic and allelic frequencies. Genotypic frequencies for C1236T (rs1128503) were 0.31 for CC, 0.60 for CT and 0.09 for TT and allelic frequencies were 0.61 and 0.39 for C and T, respectively. Genotypic frequencies for C3435T (rs1045642) were 0.24 for CC, 0.61 for CT and 0.15 for TT, and allelic frequencies were 0.55 and 0.45 for C and T, respectively. Both variants showed significant differences in genotypic frequencies for both studied regions. Gender comparisons did not show significant differences between genotypes at both sites. Significant differences were observed between C1236C and C3435T and also C1236C and C3435C. Comparisons between ABCB1 polymorphisms in Brazilians and other populations show significant differences. The understanding of the effects of several drugs associated to these variants may help an individualized treatment in clinical practice.     

CLONAL STRUCTURE OF ARCTIC DWARF BIRCH (BETULA GLANDULOSA) AT ITS NORTHERN LIMIT

Betula glandulosa Michx. (Betulaceae) at Tarr Inlet, Baffin Island, Northwest Territories reproduces primarily by vegetative layering with little, if any, viable seed production or seedling recruitment. Information from polymorphic enzyme loci revealed 15 three-locus genotypes with a single genotype usually dominating each of the nine sampled sites. Clonal reproduction combined with self-incompatibility may be one factor contributing to a decreased seed set by reducing the probability of xenogamy. However, multiple genotypes were found at some sites providing an opportunity for cross compatible pollen to effect fertilization. The lack of seed set in these areas appears to be due to additional factors inhibiting sexual reproduction. Based on pollen records, the present limited distribution of Betula glandulosa on Baffin Island represents the remnants of once more widely distributed populations, and the failure of sexual reproduction at the northern edge of its range may be responsible for the decline in abundance. Genotypic diversity observed in the Tarr Inlet area appears to be residual from a once sexually reproducing population. No evidence was found for differential selection of genotypes indicating that random processes are probably eroding the genotypic diversity during the decrease of population size in this species.     

A multiplicative-epistatic model for analyzing interspecific differences in outcrossing species

Epistasis may play an important role in evolution and speciation. Under multiplicative interactions between different loci, an analytical model is proposed to estimate genetic parameters at the individual locus level that contribute to interspecific differences in outcrossing species. The multiplicative epistasis model, inferred from a number of animal and plant experiments, suggests that genotypes at a pair of loci have genotypic values equal to the product of genotypic values at the two different loci. By considering the genetic property of outcrossing species (i.e., high polymorphisms) in the multilevel family structure analysis for an intra- and interspecific factorial mating design, a method is developed to provide estimates for allele frequencies and additive and dominant effects at individual loci in each of the two parental populations, the genotypic values of newly formed heterozygotes through species combination each with one allele from a parental population and the second from the other parental population, and the numbers of genetic factors that lead to species differentiation. Use of clones offers a tremendous power to test the adequacy of the model. However, the utilization of the model with species that cannot be cloned is also discussed. An example with interspecific hybrids of two forest tree species is used to demonstrate the model.