Electrophoretic Alleles Research Articles

Genetic markers of adaptive processes in the Far Eastern pink salmon Oncorhynchus gorbuscha: Allelic diversity at the locus of major histocompatibility complex MHC I-A1

To clarify allelic diversity at the locus of major histocompatibility complex MHC class I-A1 in the Far Eastern pink salmon Oncorhynchus gorbuscha, sequencing of the electrophoretic alleles isolated from the gel (DGGE alleles) was performed. In 47 individuals, the genotypes of which consisted of ten DGGE alleles, 18 MHC I-A1 nucleotide sequences were revealed, and thus, eight cryptic alleles not detected by electrophoresis were identified. Eleven of these alleles were identified earlier in pink salmon from Hokkaido, Alaska, and British Columbia, and seven, possibly, were unique to the populations from some Far Eastern regions. Six of the previously determined DGGE alleles corresponded to more than one nucleotide sequence. However, the sequences attributed to the same DGGE allele differed on average by less than 1 nucleotide. These findings point to sufficient sensitivity of the DGGE method, although the genetic diversity and differentiation estimates obtained with it will obviously be somewhat underestimated. Considerable predominance of nonsynonymous substitutions over the synonymous ones in the codons of the MHC I-A1 antigen-binding site confirms the presence of positive selection aimed at providing the population resistance to local spectrum of pathogens. Refinement of the allelic composition of the adaptively important MHC genetic marker will contribute to more complete understanding of the adaptive genetic structure of pink salmon as an important element of the overall population structure of the species.

Selection at the Esterase-2 locus of Drosophila buzzatii? Perturbation-reperturbation experiments.

Apparent selection affecting starch gel electrophoretic alleles at the Esterase-2 locus of Drosophila buzzatii has been detected in laboratory and natural populations. Perturbation-reperturbation of allele frequencies in replicated laboratory populations attempts to test direct selective effects at the locus versus effects of linked loci. Sequential gel electrophoresis has identified more alleles within starch classes, and three of these alleles (within the a, b and c starch alleles) were used in cage population experiments. Allele a/1.00/1.00/1.00 was set up in 10 replicate populations with allele c/1.00/1.00/1.00, and in an independent 10 replicate populations with allele b/0.99/1.01/1.00. For each set, three reperturbations were done. Replicate populations generally showed similar patterns of allele frequency change and clear directionality: effects of selection, not drift. However, four populations deviated from their replicates, indicating dissipation of linkage disequilibrium. Estimates of pre-adult viability in the F2 of pair-wise crosses among 12 sequential gel electrophoretic alleles showed very variable modes of inheritance and relative viability fitnesses. Together with the diversity of patterns of allele frequency change in the cage populations, these results suggest a gene complex, with selection acting on an interacting set of loci which may include Esterase-2.

Revisiting protein heterozygosity in plants—nucleotide diversity in allozyme coding genes of conifer Pinus sylvestris

Allozyme variation has been and continues to be a major source of information on the level of genetic variation among plant species. Deciphering the molecular basis of electrophoretic variation is essential for understanding the forces affecting the protein level variation. In this study, the relationship between allozyme heterozygosity and nucleotide diversity in plants is investigated among and within species. Allozyme and nucleotide diversity in 27 plant species was reviewed. At the multilocus level, the two methods are congruent: a clear correlation between the two measures of genetic diversity among plant species was observed, strengthening the view that effective population size is the major determinant of genome-wide diversity. Nucleotide diversity at six allozyme coding genes (6pgdB, aco, gdh, gotC, mdhA, and mdhB) in conifer Pinus sylvestris was investigated jointly with electrophoretic data. Single non-synonymous charge-changing mutations were found together with electrophoretic alleles that consequently were mutationally unique. Synonymous site nucleotide diversity (point estimate of θW—0.009 per bp) and silent site divergence from Pinus pinaster at allozyme coding loci were at comparable levels with other loci in the species. Linkage disequilibrium was extensive compared to earlier estimates from P. sylvestris and other trees, spanning several kilobases. Allozyme coding genes had an excess of closely related haplotypes whose frequency has recently increased possibly as a result of partial selective sweeps or balancing selection, but complex demographic effects cannot be excluded.

A Component of Isolation between <I>Allonemobius shalontaki</I> (Orthoptera: Gryllidae: Nemobiinae) and a Sympatric Congener

Allonemobius shalontaki (Braswell) (Orthoptera: Gryllidae: Nemobiinae), a newly recognized species, was described on the basis of novel electrophoretic alleles and a unique male calling song. However, little is known about the species and the factors that isolate it from co-occurring species of Allonemobius (Hebard). We use electrophoretic markers to document sympatry of A. shalontaki with its congener Allonemobius socius (Scudder) and to follow the life cycle of the two species at two sites. The results indicate that there are phenological differences between the species. To confirm the phenological differences quantified in the field, we study patterns of egg development and hatching in the laboratory. Together with laboratory hybridization studies, these result demonstrate that A. shalontaki and A. socius are strongly, but not completely, reproductively isolated.

Tree scanning: a method for using haplotype trees in phenotype/genotype association studies.

We use evolutionary trees of haplotypes to study phenotypic associations by exhaustively examining all possible biallelic partitions of the tree, a technique we call tree scanning. If the first scan detects significant associations, additional rounds of tree scanning are used to partition the tree into three or more allelic classes. Two worked examples are presented. The first is a reanalysis of associations between haplotypes at the Alcohol Dehydrogenase locus in Drosophila melanogaster that was previously analyzed using a nested clade analysis, a more complicated technique for using haplotype trees to detect phenotypic associations. Tree scanning and the nested clade analysis yield the same inferences when permutation testing is used with both approaches. The second example is an analysis of associations between variation in various lipid traits and genetic variation at the Apolipoprotein E (APOE) gene in three human populations. Tree scanning successfully identified phenotypic associations expected from previous analyses. Tree scanning for the most part detected more associations and provided a better biological interpretative framework than single SNP analyses. We also show how prior information can be incorporated into the tree scan by starting with the traditional three electrophoretic alleles at APOE. Tree scanning detected genetically determined phenotypic heterogeneity within all three electrophoretic allelic classes. Overall, tree scanning is a simple, powerful, and flexible method for using haplotype trees to detect phenotype/genotype associations at candidate loci.

Comparative Molecular Population Genetics of the Xdh Locus in the Cactophilic Sibling Species Drosophila buzzatii and D. koepferae

The Xdh (rosy) gene is one of the best studied in the Drosophila genus from an evolutionary viewpoint. Here we analyze nucleotide variation in a 1875-bp fragment of the second exon of Xdh in Argentinian populations of the cactophilic D. buzzatii and its sibling D. koepferae. The major electrophoretic alleles of D. buzzatii not only lack diagnostic amino acids in the region studied but also differ on average from each other by four to 13 amino acid changes. Our data also suggest that D. buzzatii populations belonging to different phytogeographic regions are not genetically differentiated, whereas D. koepferae exhibits a significant pattern of population structure. The Xdh region studied is twice as polymorphic in D. buzzatii as in D. koepferae. Differences in historical population size or in recombinational environment between species could account for the differences in the level of nucleotide variation. In both species, the Xdh region exhibits a great number of singletons, which significantly departs from the frequency spectrum expected under neutrality for nonsynonymous sites and also for synonymous sites in D. buzzatii. These excesses of singletons could be the signature of a recent population expansion in D. buzzatii, whereas they may be simply explained as the result of negative selection in D. koepferae.

The relationship between allozyme and chromosomal polymorphism inferred from nucleotide variation at the Acph-1 gene region of Drosophila subobscura.

The Acph-1 gene region was sequenced in 51 lines of Drosophila subobscura. Lines differ in their chromosomal arrangement for segment I of the O chromosome (O(st) and O(3+4)) and in the Acph-1 electrophoretic allele (Acph-1(100), Acph-1(054), and Acph-1(>100)). The ACPH-1 protein exhibits much more variation than previously detected by electrophoresis. The amino acid replacements responsible for the Acph-1(054) and Acph-1(>100) electrophoretic variants are different within O(st) and within O(3+4), which invalidates all previous studies on linkage disequilibrium between chromosomal and allozyme polymorphisms at this locus. The Acph-1(>100) allele within O(3+4) has a recent origin, while both Acph-1(054) alleles are rather old. Levels of nucleotide variation are higher within the O(3+4) than within the O(st) arrangement except for nonsynonymous sites. The McDonald and Kreitman test shows a significant excess of nonsynonymous polymorphisms within O(st) when D. guanche is used as the outgroup. According to the nearly neutral model of molecular evolution, this excess is consistent with a smaller effective size of O(st) relative to O(3+4) arrangements. A smaller population size, a lower recombination, and a more recent bottleneck might be contributing to the smaller effective size of O(st).

Genetic variability in biochemical characters of Brazilian field populations of the Leishmania vector, Lutzomyia longipalpis (Diptera: Psychodidae).

The phlebotomine sand fly Lutzomyia longipalpis is the insect vector of visceral leishmaniasis, a protozoan disease of increasing incidence and distribution in Central and South America. Electrophoretic allele frequencies of 15 enzyme loci were compared among the L. longipalpis populations selected across its distribution range in Brazil. The mean heterozygosity of two colonized geographic strains (one each from Colombia and Brazil) were 6% and 13% respectively, with 1.6-1.9 alleles detected per locus. In contrast, among the seven widely separated field populations, the mean heterozygosity ranged from 11% to 16% with 2.1-2.9 alleles per locus. No locus was recovered that was diagnostic for any of the field populations. Allelic frequency differences among five field strains from the Amazon basin and eastern coastal Brazil were very low, with Nei's genetic distances of less than 0.01 separating them. The two inland and southerly samples from Minas Gerais (Lapinha) and Bahia (Jacobina) states were more distinctive with genetic distances of 0.024-0.038 and 0.038-0.059, respectively, when compared with the five other samples. These differences were the consequence of several high frequency alleles (glycerol-3-phosphate dehydrogenase [Gpd1.69] and phosphoglucomutase [Pgm1.69]) relatively uncommon in other strains. The low genetic distances, absence of diagnostic loci, and the distribution of genes in geographic space indicate L. longipalpis of Brazil to be a single, but genetically heterogeneous, polymorphic species.

Molecular structure of rare but geographically widespread sn-glycerol-3-phosphate dehydrogenase 'ultra-fast' electrophoretic alleles in Drosophila melanogaster.

Six naturally occurring but rare alleles of sn-glycerol-3-phosphate dehydrogenase (Gpdh) in Drosophila melanogaster have been investigated in this study. They all belong to a class of GpdhUF (ultra-fast) alleles, because their electrophoretic mobilities are faster than that of the GpdhF (fast) allele. The GpdhUF variants are widespread, and have been reported from five continents. DNA sequence analysis has shown that the change in electrophoretic mobility was in each allele caused by a single amino acid residue substitution in the encoded protein. In the XiamenUF allele it is a substitution of lysine (AAA) to asparagine (AAT) in exon 1 (residue 3). An asparagine (AAT) to aspartate (GAT) change was found in exon 6 (residue 336) in the IowaUF and NetherlandsUF alleles. The mobility of the RaleighUF allele was altered by a valine (GTG) to glutamate (GAG) substitution in exon 3 (residue 76). Two mutations were detected in the BrazzavilleUF allele: a lysine (AAG) to methionine (ATG) substitution in exon 2 (residue 68) is responsible for the ultra-fast phenotype of this variant, while a tyrosine (TAT) to phenylalanine (TTT) substitution in exon 4 (residue 244) is not expected to alter the electrophoretic mobility of the encoded protein. These results indicate that the GpdhUF alleles originate from different mutational events, and only two of them--IowaUF and NetherlandsUF--might share a common ancestry. The GPDH activity of the IowaUF allele is intermediate between those of the GpdhS and GpdhF control stocks. The other GpdhUF variants have lower activities than the controls: XiamenUF--83%, RaleighUF--80% and BrazzavilleUF--73% of the GpdhF control.

Molecular analysis of a Drosophila melanogaster sn-glycerol-3-phosphate dehydrogenase allozyme variant that has cold labile activity

A rare naturally occurring allele, Gpdh ACb62 , at the sn-glycerol-3-phosphate dehydrogenase locus in Drosophila melanogaster encodes an enzyme with an electrophoretic mobility that is more cathodal than that produced by the common slow electrophoretic allele. After electrophoresis and staining of extracts of single adult flies there is a single band of activity corresponding in position to GPDH-1, but, using highly concentrated extracts, a faint band corresponding to GPDH-3 is observed. In Gpdh ACb62 homozygotes there is about 26% of the normal level of activity in adults, and less than 6% in third instar larvae. The reduction in activity is significantly greater than the decrease in GPDH immunologically cross-reacting material (CRM). Northern analyses, and rapid amplification of the cDNA ends (RACE) of the 3' regions of the transcripts, show that the levels and structures of the poly(A) +RNAs are similar in homozygotes for Gpdh ACb62 and for a normal activity allele Gpdh AC8 . Hybridization to oligonucleotide probes specific for the GPDH-1 and GPDH-3 transcripts was of a similar intensity in Gpdh ACb62 and Gpdh ACb62 adult flies. In third instar larvae the main transcript is for GPDH-3 and again the hybridization signals were similar in each line. The activity of the enzyme produced by Gpdh ACb62 was unstable both at 50°C and at 0°C. The activity lost at 0°C was recovered by incubation at 20°C. The complete Gpdh ACb62 gene, and the partial Gpdh tandem duplication 3' to this gene, were cloned and sequenced. Comparisons with two normal activity Gpdh f genes revealed 31 unique changes in the first copy of Gpdh ACb62 . In exon 4, a T to G substitution changes cysteine to glycine and may disrupt a disulphide bond and be responsible for the distinctive properties of GPDH-ACb62.

Genetic Diversity of Feral Honey Bee (Hymenoptera: Apidae) Populations in the Southern United States

Mitochondrial DNA (mtDNA) and allozyme analyses of 692 feral honey bee, Apis mellifera L., colonies revealed significant genetic population structure (heterogeneity) in the southern tier of the United States. Analysis of mtDNA haplotypes showed that 61% of the colonies were maternal descendants of the European races most commonly used for commercial beekeeping and 37% were descendants of a race ( A. m. mellifera L.) that was imported between 1620 and the 1860s, but is no longer used commercially. Twelve colonies from five states exhibited an African haplotype associated with the race A. m. lamarckii Cockerell introduced from Egypt >100 yr ago. Several electrophoretic alleles not previously reported from U.S. populations were detected, and malate dehydrogenase allele frequencies for the 692 colonies were determined to be Mdh65 = 0.47, Mdh80 = 0.33, and Mdh100 = 0.20. Despite the significant heterogeneity of allele frequencies, no significant deviations from Hardy-Weinberg expectations were detected when all bees sampled were artificially pooled into one population or by individual states. The Mdh80 frequency was higher than that found in previous studies of commercial populations, suggesting a disproportionately higher genetic contribution of A. m. mellifera to the feral population. The discovery of significant heterogeneity among U.S. feral honey bees implies that this population may represent a source of genetic variation for breeding programs.

Haplotypic divergence coupled with lack of diversity at the Arabidopsis thaliana alcohol dehydrogenase locus: roles for both balancing and directional selection?

We designate a region of the alcohol dehydrogenase locus (Adh) of the weedy crucifer, Arabidopsis thaliana, as "hypervariable" on the basis of a comparison of sequences from ecotypes Columbia and Landsberg. We found eight synonymous and two replacement mutations in the first 262 nucleotides of exon 4, and an additional two mutations in the contiguous region of intron 3. The rest of the sequence (2611 bp) has just three mutations, all of them confined to noncoding regions. Our survey of the hypervariable region among 37 ecotypes of A. thaliana revealed two predominant haplotypes, corresponding to the Columbia and Landsberg sequences. We identified five additional haplotypes and 4 additional segregating sites. The lack of haplotype diversity is presumably in part a function of low rates of recombination between haplotypes conferred by A. thaliana's tendency to self-fertilize. However, an analysis in 32 ecotypes of 12 genome-wide polymorphic markers distinguishing Columbia and Landsberg ecotypes indicated levels of outcrossing sufficient at least to erode linkage disequilibrium between dispersed markers. We discuss possible evolutionary explanations for the coupled observation of marked divergence within the hypervariable region and a lack of haplotype diversity among ecotypes. The sequence of the region for closely related species argues against the possibility that one allele is the product of introgression. We note (1) that several loss of function mutations (both naturally and chemically induced) map to the hypervariable region, and (2) the presence of two amino acid replacement polymorphisms, one of which causes the mobility difference between the two major classes of A. thaliana Adh electrophoretic alleles. We argue that protein polymorphism in such a functionally significant part of the molecule may be subject to balancing selection. The observed pattern of extensive divergence between the alleles is consistent with this explanation because balancing selection on a particular site maintains linked neutral polymorphisms at intermediate frequencies.

Clinal variation and selection of MDH allozymes in honey bee populations.

Latitudinal clines of malate dehydrogenase-1 (MDH-1) allozymes occur within honey bee populations on three continents: Europe, North America and South America. The North and South American populations are introduced and demonstrate that Mdh allelic clines were established within the last 150 years. The frequency of the 'medium' electrophoretic allele increases in frequency with increasing latitude while the 'fast' allele decreases with latitude on all the three continents. The clines are best explained by the average daily high temperature for July on all continents. These parallel clines provide evidence for selection on Mdh alleles in honey bees.

House Mice as Models in Systematic Biology

We evaluated the house mouse (Mus musculus species complex) and its laboratory descendents as models for three aspects of systematic biology: hybrid zone biology, chromosomal evolution and speciation, and tests for methods of phylogenetic reconstruction. The taxonomy and genetic relationships of the species in the complex are summarized. The comparative phylogenetic method was used to illucidate biogeographical, ecological, and chromosomal events in the group. Hybridization in house mice was evaluated with respect to the emerging discipline of hybrid zone biology. Inter- and intraspecific contact zones are described and compared. Zonespecific electrophoretic alleles, new metacentric chromosomes, and increased levels of parasitism are explained as consequences of genome disruption. The interspecific zone is widest in the area of most recent contact between species. Variation in the widths of individual clines is discussed. Chromosomal evolution is proceeding along alternative paths in different commensal lineages of house mice. A karyotypic revolution occurred within the species M. domesticus that led to the reproductive isolation between two local populations. The recency of this karyotypic revolution permits a study of how Robertsonian populations evolve and an evaluation of whether the stasipatric model of speciation is a good explanation for this case. The genealogical history of the inbred strains of mice is well known, and this information has been used to test how faithfully different kinds of data and different kinds of analytical techniques recreate the known phylogeny. Molecular data recapture the correct phylogeny better than do morphological data, and the commonly used analytical methods are all equally robust in producing this phylogeny.

House Mice as Models in Systematic Biology

We evaluated the house mouse (Mus musculus species complex) and its laboratory descendents as models for three aspects of systematic biology: hybrid zone biology, chromosomal evolution and speciation, and tests for methods of phylogenetic reconstruction. The taxonomy and genetic relationships of the species in the complex are summarized. The comparative phylogenetic method was used to illucidate biogeographical, ecological, and chromosomal events in the group. Hybridization in house mice was evaluated with respect to the emerging discipline of hybrid zone biology. Inter- and intraspecific contact zones are described and compared. Zone- specific electrophoretic alleles, new metacentric chromosomes, and increased levels of parasitism are explained as consequences of genome disruption

The effect of superoxide dismutase alleles on aging in Drosophila.

The effects of superoxide dismutase on aging were tested using two different experimental approaches. In the first, replicated populations with postponed aging were compared with their controls for frequencies of electrophoretic alleles at the SOD locus. Populations with postponed aging had consistently greater frequencies of the allele coding for more active SOD protein. This allele was not part of a segregating inversion polymorphism. The second experimental approach was the extraction of SOD alleles from different natural populations followed by the construction of different SOD genotypes on hybrid genetic backgrounds. This procedure did not uncover any statistical effect of SOD genotype on longevity or fecundity. There were large effects on longevity and fecundity due to the family from which a particular SOD genotype was derived. To detect the effects of SOD genotypes on longevity with high probability would require a ten-fold increase in the number of families used.

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products.

Two, electrophoretically distinct, forms of carbonic anhydrase I (CA Ia and CA Ib) are found at high polymorphic frequencies in red cells of natural populations of pigtail macaques, Macaca nemestrina, from southeast Asia. By use of the polymerase chain reaction, exons of the CA I gene were amplified from homozygous (a/a, b/b) and heterozygous (a/b) animals. Direct sequencing of the amplified DNA from four animals revealed differences between the a and the b electrophoretic alleles ranging from three to six nucleotides, and from one to three differences within each allele. These results indicate a greater genetic variability at the CA I locus in this macaque species than previously realized.

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products

Two, electrophoretically distinct, forms of carbonic anhydrase I (CA Ia and CA Ib) are found at high polymorphic frequencies in red cells of natural populations of pigtail macaques,Macaca nemestrina, from southeast Asia. By use of the polymerase chain reaction, exons of the CA I gene were amplified from homozygous (a/a, b/b) and heterozygous (a/b) animals. Direct sequencing of the amplified DNA from four animals revealed differences between the a and the b electrophoretic alleles ranging from three to six nucleotides, and from one to three differences within each allele. These results indicate a greater genetic variability at the CA I locus in this macaque species than previously realized.

Evolution of Lake Whitefish (Coregonus clupeaformis) in North America during the Pleistocene: Evidence for a Nahanni Glacial Refuge Race in the Northern Cordillera Region

Lake whitefish (Coregonus clupeaformis) could have survived through (at least) the Illinoisan and Wisconsinan Pleistocene glacial maxima in an area in the vicinity of the present Nahanni National Park in the Northwest Territories of Canada according to the geological evidence. This possibility was addressed by an analysis of the genetic makeup of 43 lake whitefish populations in the Northwest Territories, the Yukon Territory, British Columbia, and Alberta. Populations in the lower Liard, Tetcela, Fraser, and upper Peace River systems as well as the headwaters of the Athabasca River were distinguished from both the Bering glacial refuge race populations inhabiting the Yukon and upper Liard River basins in the Yukon Territory and the Mississippi–Missouri glacial refuge race populations inhabiting most of the Northwest Territories, Alberta, and areas further to the east by a specific combination of electrophoretic mobility alleles. This evidence supports the hypothesis of the survival and subsequent dispersal of lake whitefish from a Nahanni glacial refugium. Possible dispersal routes and the limited extent of introgression among races are discussed.