GENETIC LOAD IN OSMUNDA REGALIS POPULATIONS

Osmunda regalis sporophytes form haploid spores which develop into functionally hermaphroditic gametophytes. The self-fertilization of such gametophytes results in zygotes which are completely homozygous. Spore samples collected from sporophytes in natural populations were used to establish gametophyte cultures. The majority of these gametophytes were unable to form viable embryos when only self-fertilization was possible. Controlled selfing and crossing experiments revealed that the inability of these homozygous embryos to develop normally is attributable to the presence of recessive lethals. To account for this genetic load, an hypothesis is proposed integrating the morphology and ecology of the gametophyte generation with the polyploid genetic system of the sporophyte generation.     

EVIDENCE FOR GENETIC HETEROZYGOSITY IN A HOMOSPOROUS FERN

Homosporous fern sporophytes from natural populations exhibited heterozygous electrophoretic patterns for several enzyme systems. Genetic tests utilizing individual gametophytes demonstrate that the observed heterozygosity is coded by alleles at single loci. This simple procedure makes it possible to distinguish segregating from fixed or phenotypic heterozygosity, previously a problem in homosporous vascular plants.     

Marker-assisted genotype analysis of bulb colors in segregating populations of onions (Allium cepa)

Bulb color in onions (Allium cepa) is an important trait whose complex inheritance mechanism involves epistatic interactions among major color-related loci. Recent studies revealed that inactivation of dihydroflavonol 4-reductase (DFR) in the anthocyanin synthesis pathway was responsible for the color differences between yellow and red onions, and two recessive alleles of the anthocyanidin synthase (ANS) gene were responsible for a pink bulb color. Based on mutations in the recessive alleles of these two genes, PCR-based markers for allelic selection were developed. In this study, genotype analysis of onions from segregating populations was carried out using these PCR-based markers. Segregating populations were derived from the cross between yellow and red onions. Five yellow and thirteen pink bulbs from one segregating breeding line were genotyped for the two genes. Four pink bulbs were heterozygous for the DFR gene, which explains the continuous segregation of yellow and pink colors in this line. Most pink onions were homozygous recessive for the ANS gene, except for two heterozygotes. This finding indicated that the homozygous recessive ANS gene was primarily responsible for the pink color in this line. The two pink onions, heterozygous for the ANS gene, were also heterozygous for the DFR gene, which indicated that the pink color was produced by incomplete dominance of a red color gene over that of yellow. One pink line and six other segregating breeding lines were also analyzed. The genotyping results matched perfectly with phenotypic color segregation.     

OBLIGATE OUTCROSSING IN A HOMOSPOROUS FERN: FIELD CONFIRMATION OF A LABORATORY PREDICTION

While homosporous ferns are potentially capable of producing totally homozygous sporophytes in one generation via selfing of their bisexual gametophytes, laboratory analyses indicate that a variety of mechanisms promote gametophytic outcrossing. The operation of these mechanisms in natural sporophyte populations, however, has not been previously demonstrated. Laboratory analyses of gametophyte ontogeny show that Bommeria hispida is obligately outcrossing. Electrophoretic data presented here indicate that individuals from natural sporophyte populations of this species are highly heterozygous. Electrophoretic data, therefore, corroborate evidence from the in vitro analysis of gametophyte development and demonstrate that sporophytes of B. hispida in nature typically are products of outcrossing between genetically different gametophytes. Extrapolations from the literature, together with our findings, indicate that outcrossing mechanisms may operate frequently in ferns, thereby maintaining genetic variability between individuals within populations. This evidence questions whether most ferns are highly inbred and therefore predominantly homozygous.     

X‐AUTOSOME INCOMPATIBILITIES IN DROSOPHILA MELANOGASTER: TESTS OF HALDANE'S RULE AND GEOGRAPHIC PATTERNS WITHIN SPECIES

Substantial genetic variation exists in natural populations of Drosophila melanogaster. This segregating variation includes alleles at different loci that interact to cause lethality or sterility (synthetic incompatibilities). Fitness epistasis in natural populations has important implications for speciation and the rate of adaptive evolution. To assess the prevalence of epistatic fitness interactions, we placed naturally occurring X chromosomes into genetic backgrounds derived from different geographic locations. Considerable amounts of synthetic incompatibilities were observed between X chromosomes and autosomes: greater than 44% of all combinations were either lethal or sterile. Sex‐specific lethality and sterility were also tested to determine whether Haldane's rule holds for within‐species variation. Surprisingly, we observed an excess of female sterility in genotypes that were homozygous, but not heterozygous, for the X chromosome. The recessive nature of these incompatibilities is similar to that predicted for incompatibilities underlying Haldane's rule. Our study also found higher levels of sterility and lethality for genomes that contain chromosomes from different geographical regions. These findings are consistent with the view that genomes are coadapted gene complexes and that geography affects the likelihood of epistatic fitness interactions.     

Inbreeding load, average dominance and the mutation rate for mildly deleterious alleles in Mimulus guttatus

The goal of this study is to provide information on the genetics of inbreeding depression in a primarily outcrossing population of Mimulus guttatus. Previous studies of this population indicate that there is tremendous inbreeding depression for nearly every fitness component and that almost all of this inbreeding depression is due to mildly deleterious alleles rather than recessive lethals or steriles. In this article I assayed the homozygous and heterozygous fitnesses of 184 highly inbred lines extracted from a natural population. Natural selection during the five generations of selfing involved in line formation essentially eliminated major deleterious alleles but was ineffective in purging alleles with minor fitness effects and did not appreciably diminish overall levels of inbreeding depression. Estimates of the average degree of dominance of these mildly deleterious alleles, obtained from the regression of heterozygous fitness on the sum of parental homozygous fitness, indicate that the detrimental alleles are partially recessive for most fitness traits, with h approximately 0.15 for cumulative measures of fitness. The inbreeding load, B, for total fitness is approximately 1.0 in this experiment. These results are consistent with the hypothesis that spontaneous mildly deleterious mutations occur at a rate >0.1 mutation per genome per generation.     

A general model to explore complex dominance patterns in plant sporophytic self-incompatibility systems

We developed a general model of sporophytic self-incompatibility under negative frequency-dependent selection allowing complex patterns of dominance among alleles. We used this model deterministically to investigate the effects on equilibrium allelic frequencies of the number of dominance classes, the number of alleles per dominance class, the asymmetry in dominance expression between pollen and pistil, and whether selection acts on male fitness only or both on male and on female fitnesses. We show that the so-called "recessive effect" occurs under a wide variety of situations. We found emerging properties of finite population models with several alleles per dominance class such as that higher numbers of alleles are maintained in more dominant classes and that the number of dominance classes can evolve. We also investigated the occurrence of homozygous genotypes and found that substantial proportions of those can occur for the most recessive alleles. We used the model for two species with complex dominance patterns to test whether allelic frequencies in natural populations are in agreement with the distribution predicted by our model. We suggest that the model can be used to test explicitly for additional, allele-specific, selective forces.     

Apparent overdominance of enzyme specific activity in two marine bivalves

Electrophoretic examination of a natural population sample of 332 mussels (Mytilus trossulus) revealed ten active allozyme alleles for the octopine dehydrogenase (Odh) locus and a statistically significant (P<0.005) departure from expected genotypic proportions caused by a deficiency of heterozygous genotypes. In vitro specific activity for octopine dehydrogenase (E.C. 1.5.1.11) was determined for 207 mussels representing 17 different Odh genotypes. Odh heterozygotes had an average specific activity that was 19% greater than that of apparently homozygous genotypes, a significant (P<0.05) difference. Electrophoretic examination of a natural population sample of 209 oysters (Crassostrea virginica) revealed 23 active allozyme alleles for the leucine aminopeptidase-2 (Lap-2) locus and a non-significant (P>0.05) deficiency of heterozygous genotypes. In vitro specific activity for leucine aminopeptidase (E.C. 3.4.-.-) was determined for 89 oysters representing 19 different Lap-2 genotypes. Lap-2 heterozygotes had an average specific activity that was 56% greater than that of homozygous genotypes, a significant (P<0.0001) difference. Possible explanations for the apparent overdominance in enzyme specific activity and the deficiency of heterozygotes include null alleles, molecular imprinting and aneuploidy.     

Latitudinal variation in wing size in Drosophila subobscura and its dependence on polygenes of chromosome O

The genetic variability of natural populations of Drosophila subobscura derived from localities situated approximately along a north-to-south diameter through the distribution area of the species was studied. The polygenic and adaptive trait ‘wing size’ shows a continuous clinal reduction from north to south. The comparison between flies homozygous or randomly heterozygous for wild chromosomes showed that heterozygous flies are on an average bigger than homozygous flies and that overdominance exists for increased wing size. The genetic variance proved lower in central than in ecologically marginal populations. From the analysis of left-right asymmetry it could be shown that heterozygous flies are more homeostatic than homozygous flies but no differences appeared between populations. Therefore, lower phenotypic variability in central populations must be due to lower genetic variation. An hypothesis was put forward that normalizing selection eliminates + and-alleles in the central populations; in marginal populations, on the other hand, either + (north) or-(south) alleles are favoured and the genetic variability is augmented.     

Heterosis of Quantitative Trait Loci Affecting Lifespan in Drosophila melanogaster

Knowledge of genes responsible for aging and death is a prerequisite for determining the relative contributions of the different evolutionary factors responsible for the limited duration of life. Polymorphism of these genes probably accounts for the variation in lifespan. Previously, quantitative trait loci (QTLs) controlling this variation were mapped with the use of 98 recombinant inbred (RI) lines originating from two parental isogenicDrosophila melanogaster stocks. In each RI line, lifespan was measured for 25 males and 25 females, and alleles were established for 93 marker genes segregating between the parental lines. Significant correlation between marker segregation and lifespan was revealed for several chromosome regions. The lifespan genes had sex-specific effects and late age onset. In the present work, the effects of the QTLs were compared for homozygous and heterozygous flies. In Six out of the eight detected QTLs alleles that decreased lifespan were recessive. Heterosis was observed for a of QTL at 33E–38A. Thus, heterosis might contribute to maintaining variation in lifespan in natural populations.     

Heterosis of quantitative trait loci modifying lifespan in Drosophila melanogaster

Knowledge of genes responsible for aging and death is a prerequisite for determining the relative contributions of the different evolutionary factors responsible for the limited duration of life. Polymorphism of these genes probably accounts for the variation in lifespan. Previously, quantitative trait loci (QTLs) controlling this variation were mapped with the use of 98 recombinant inbred (RI) lines originating from two parental isogenic Drosophila melanogaster stocks. In each RI line, lifespan was measured for 25 males and 25 females, and alleles were established for 93 marker genes segregating between the parental lines. Significant correlation between marker segregation and lifespan was revealed for several chromosome regions. The lifespan genes had sex-specific effects and late age onset. In the present work, the effects of the QTLs were compared for homozygous and heterozygous flies. In Six out of the eight detected QTLs alleles that decreased lifespan were recessive. Heterosis was observed for a of QTL at 33E-38A. Thus, heterosis might contribute to maintaining variation in lifespan in natural populations.     

Mitotic recombination counteracts the benefits of genetic segregation

The ubiquity of sexual reproduction despite its cost has lead to an extensive body of research on the evolution and maintenance of sexual reproduction. Previous work has suggested that sexual reproduction can substantially speed up the rate of adaptation in diploid populations, because sexual populations are able to produce the fittest homozygous genotype by segregation and mating of heterozygous individuals. In contrast, asexual populations must wait for two rare mutational events, one producing a heterozygous carrier and the second converting a heterozygous to a homozygous carrier, before a beneficial mutation can become fixed. By avoiding this additional waiting time, it was shown that the benefits of segregation could overcome a twofold cost of sex. This previous result ignores mitotic recombination (MR), however. Here, we show that MR significantly hastens the spread of beneficial mutations in asexual populations. Indeed, given empirical data on MR, we find that adaptation in asexual populations proceeds as fast as that in sexual populations, especially when beneficial alleles are partially recessive. We conclude that asexual populations can gain most of the benefit of segregation through MR while avoiding the costs associated with sexual reproduction.     

Associative overdominance: Evaluating the effects of inbreeding and linkage disequilibrium

Expressions are obtained for the expected phenotypic values of homozygous and heterozygous genotypes for a neutral marker locus linked to a locus segregating for a recessive deleterious gene. The phenotypic values are functions of the allele frequencies at the marker locus, the inbreeding coefficient and the degree of association of the deleterious gene with the marker alleles. The analysis is extended to more than two alleles at the marker locus. Either linkage disequilibrium or inbreeding alone can produce an apparent superiority of heterozygotes for the marker locus (unless specified otherwise, the terms ‘homozygote’ and ‘heterozygote’ will refer to the marker locus). The effect of linkage disequilibrium on the difference between the heterozygote and homozygote values can be positive (associative overdominance) or negative (associative underdominance), depending on the frequencies of the marker alleles and the degree of their association with the deleterious gene. Inbreeding has always a positive effect. In general, the expected value of a homozygote is a positive function of its allele frequency. When the various homozygous genotypes are combined into one class and the various heterozygous genotypes into another, the phenotypic difference of the two classes is a function of the evenness of the allelic frequency distribution. Inbreeding is a more likely explanation of associative overdominance if the frequency of the deleterious gene is low, but its effect on the character high. Conversely, linkage disequilibrium is more likely if the frequency is high and the effect low. The degrees of association between marker alleles and the deleterious gene can, in principle, be estimated from the observed phenotypic scores and used to calculate expected multi-locus genotype scores. This could provide the basis for statistical tests of the associative overdominance hypothesis as an explanation of observed correlations between multi-locus heterozygosity and phenotypic traits.     

Environmental dependence of inbreeding depression and purging in Drosophila melanogaster

Elimination or reduction of inbreeding depression by natural selection at the contributing loci (purging) has been hypothesized to effectively mitigate the negative effects of inbreeding in small isolated populations. This may, however, only be valid when the environmental conditions are relatively constant. We tested this assumption using Drosophila melanogaster as a model organism. By means of chromosome balancers, chromosomes were sampled from a wild population and their viability was estimated in both homozygous and heterozygous conditions in a favourable environment. Around 50% of the chromosomes were found to carry a lethal or sublethal mutation, which upon inbreeding would cause a considerable amount of inbreeding depression. These detrimentals were artificially purged by selecting only chromosomes that in homozygous condition had a viability comparable to that of the heterozygotes (quasi-normals), thereby removing most deleterious recessive alleles. Next, these quasi-normals were tested both for egg-to-adult viability and for total fitness under different environmental stress conditions: high-temperature stress, DDT stress, ethanol stress, and crowding. Under these altered stressful conditions, particularly for high temperature and DDT, novel recessive deleterious effects were expressed that were not apparent under control conditions. Some of these chromosomes were even found to carry lethal or near-lethal mutations under stress. Compared with heterozygotes, homozygotes showed on average 25% additional reduction in total fitness. Our results show that, except for mutations that affect fitness under all environmental conditions, inbreeding depression may be due to different loci in different environments. Hence purging of deleterious recessive alleles can be effective only for the particular environment in which the purging occurred, because additional load will become expressed under changing environmental conditions. These results not only indicate that inbreeding depression is environment dependent, but also that inbreeding depression may become more severe under changing stressful conditions. These observations have significant consequences for conservation biology.     

Biological parameters and the segregation distortion (SD) phenomenon in Drosophila melanogaster

The relationship between some biological parameters (mortality, longevity, fertility, fecundity and sex ratio) and segregation of second chromosomes in heterozygous and homozygous SD males has been analyzed. The results obtained in SD/SD+ heterozygous males show: (1) their reduced fertility with respect to that of control males, (2) an alteration in the sex ratio in the SD+ progeny only, and (3) inversely related sex-ratio and segregation distortion values. In SDi/SDj combinations: (1) surprisingly, fertility is intermediate between that of SD/SD+ heterozygous males and that of control males, (2) the segregation ratios of the second chromosomes are normal (0.50), and (3) the sex ratio = 0.50 in both classes of SD progeny. The relationship between mortality (and therefore longevity) and fertility of the different genotypes and fecundity per male indicates that the total productivity of heterozygous males is less than that so far claimed. Indeed, their productivity depends not only on the mechanism of nonformation of the SD+ sperm, but also on their reduced longevity. The k = 0.50 and the high fecundity of SDi/SDj combinations indicated that in these males the SD phenomenon is partially suppressed, the SD chromosomes being insensitive to each other, thus implying that particular Rsp alleles are sensitive to given Sd alleles. The complementation pattern for male fertility of SD homozygous males again supports previous evidence that Sd factors from natural populations are, in effect, different Sd genes.     

Genotypic Characterization of an MHC Class II Locus in Lake Trout (Salvelinus namaycush) from Lake Superior by Single-Stranded Conformational Polymorphism Analysis and Reference Strand–Mediated Conformational Analysis

This study compares the genotypic information provided by reference strand–mediated conformational analysis and single-stranded confirmational polymorphism (SSCP) analysis for the major histocompatibility complex (MHC) II locus in lake trout. For this study 80 wild-caught animals from the Apostle Islands of Lake Superior were genotyped using both RSCA and SSCP analysis. Their genotypes were recorded using both methods and compared. The genotypic information provided by the 2 methods was essentially the same although some inconsistencies were observed. Both methods detected approximately 65 genotypes, and both were able to distinguish heterozygous and homozygous animals. The analyses determined that only approximately 20% of alleles were shared between 2 morphologically different populations within the sample set, and identified the dominant alleles. SSCP analysis was quicker, simple, and more robust than RSCA. SSCP analysis using fluorescence technologies could be the method of choice for future genotypic analysis of the MHC II locus in salmonids.     

Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas' disease in Argentina

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.     

Comparison of amplification refractory mutation system and polymerase chain reaction-restriction fragment length polymorphism techniques used for the investigation of MEFV gene exon 10 point mutations in familial Mediterranean fever patients living in Cukurova region (Turkey)

Familial Mediterranean fever (FMF) is an autosomal recessive inherited disease characterized by recurrent fever, serositis and arthritis. The disease is highly prevalent in Mediterranean basin populations. Recently, the gene responsible for FMF (MEFV) was cloned and at least 40 MEFV gene mutations have been identified. The most frequently observed mutations in the MEFV gene are M694V, M694I, M680I, and V726A. These occur within exon 10 of the gene, and account for 85% of the known MEFV alleles. In this study, the reliability and economical aspects of amplification refractory mutation system (ARMS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques were compared for analyzing the frequencies of the major point mutations of 90 unrelated patients with FMF from the Cukurova region in Turkey. Both techniques yielded similar results: The ratio of independent alleles of 90 patients carrying one of the tested mutations was 81.1%; patients consisted of 12 different genotypes. In 64 of 90 patients (71.1%) mutations were observed in both alleles. Thirty-six patients (40%) were homozygous for the same mutation, 28 (31.1%) were heterozygous for different mutations. Eighteen patients (20%) were heterozygous for one allele with one of the four mutations but the other allele was unknown. In 8 patients (8.8%) no mutation could be detected. The most frequently observed mutation was M694V (51.66%), followed by M680I (17.22%), V726A (10.55%), and M694I (1.66%). In conclusion ARMS and PCR-RFLP techniques were equally reliable to detect the mutations in Turkish FMF patients. However, the ARMS technique was found to be more rapid and economical than the PCR-RFLP techniques.     

Diploid Gynogenesis in the Mexican Axolotl

Gynogenetic diploid axolotls were produced by activating eggs with ultraviolet-inactivated sperm, and then subjecting the activated eggs to heat shock. Optimal conditions for ultraviolet inactivation of the sperm, and for suppression of the second meiotic division by heat shock, were established. Gynogenetic diploids produced by these procedures included progeny homozygous for recessive alleles carried by a heterozygous mother. Gynogenesis could, therefore, be used to uncover new mutations more rapidly than by conventional inbreeding techniques. However, some difficulty was encountered in recognizing mutant phenotypes because of the high incidence of abnormalities and deaths. Defective embryos probably resulted from a combination of heat-shock-induced damage to the eggs and the expression of deleterious recessive alleles carried by the mother.     

The incidence of the various genotypes in a population showing a sex-linked recessive character

Summary Although the inheritance of a sex-linked recessive trait is well known, not all consequences are sufficiently realised. Therefore, the facts are presented once more in a new form.All populations that are unaffected by selection tend to stabilisation. When this has been reached the five different genotypes are bound by definite and rather simple numerical relations. Whereas the percentages of normal (X-) and affected (x-) males, and also those of homozygous normal (XX) and affected (xx) females may vary between 0 and 100, the percentage of heterozygous females (Xx) in a stable population never exceeds 50%.