Genetic architecture of a morphological shape difference between two Drosophila species.

The size and shape of the posterior lobe of the male genital arch differs dramatically between Drosophila simulans and D. mauritiana. This difference can be quantified with a morphometric descriptor (PC1) based on elliptical Fourier and principal components analyses. The genetic basis of the interspecific difference in PC1 was investigated by the application of quantitative trait locus (QTL) mapping procedures to segregating backcross populations. The parental difference (35 environmental standard deviations) and the heritability of PC1 in backcross populations (>90%) are both very large. The use of multiple interval mapping gives evidence for 19 different QTL. The greatest additive effect estimate accounts for 11. 4% of the parental difference but could represent multiple closely linked QTL. Dominance parameter estimates vary among loci from essentially no dominance to complete dominance, and mauritiana alleles tend to be dominant over simulans alleles. Epistasis appears to be relatively unimportant as a source of variation. All but one of the additive effect estimates have the same sign, which means that one species has nearly all plus alleles and the other nearly all minus alleles. This result is unexpected under many evolutionary scenarios and suggests a history of strong directional selection acting on the posterior lobe.

Molecular dissection of a major gene effect on a quantitative trait: the level of alcohol dehydrogenase expression in Drosophila melanogaster.

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce "superalleles" with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect.

Genetic analysis of a morphological shape difference in the male genitalia of Drosophila simulans and D. mauritiana.

Two closely related species of Drosophila, D. simulans and D. mauritiana, differ markedly in morphology of the posterior lobe of the male genital arch. Both size and shape aspects of lobe variation can be quantified by a morphometric descriptor based on elliptical Fourier and principal components analyses. The genetic architecture of this quantitative trait (PC1) was investigated by hybridizing inbred lines to produce two backcross populations approximately 200 individuals each, which were analyzed jointly by a composite interval mapping procedure with the aid of 18 marker loci. The parental lines show a large difference in PC1 (30.4 environmental standard deviations), and the markers account for > 80% of the phenotypic variation in backcross populations. Eight of 15 intervals analyzed show convincing evidence of quantitative trait loci (QTL), and the range of estimated QTL effects is 5.7-15.9% of the parental difference (1.7-4.8 environmental standard deviations). These estimates may represent the joint effects of multiple QTL within a single interval (which averaged 23 cM in length). Although there is some evidence of partial dominance of mauritiana alleles and for epistasis, the pattern of inheritance is largely additive.

The effect of an intronic polymorphism on alcohol dehydrogenase expression in Drosophila melanogaster.

Several lines of evidence indicate that natural selection controls the frequencies of an allozyme polymorphism at the alcohol dehydrogenase (Adh) locus in Drosophila melanogaster. However, because of associations among sequence polymorphisms in the Adh region, it is not clear whether selection acts directly (or solely) on the allozymic site. This problem has been approached by using in vitro mutagenesis to distinguish among the effects on Adh expression of individual polymorphisms. This study shows that a polymorphism within the first Adh intron (delta 1) has a significant effect on the level of ADH protein. Like the allozyme, delta 1 shows a geographic cline in frequency, indicating that it may also be a target of natural selection. These results suggest that multisite selection models may be required to understand the evolutionary dynamics of individual loci.

Quantitative analysis of RNA produced by slow and fast alleles of Adh in Drosophila melanogaster.

The alcohol dehydrogenase (ADH) locus (Adh) of Drosophila melanogaster in polymorphic on a world-wide basis for two allozymes, Fast and Slow. This study was undertaken to determine whether the well-established difference in ADH protein concentration between the allozymes is due to a difference in mRNA levels. RNA gel blot hybridization and an RNase protection assay were used to quantify ADH mRNA levels. Each method used an Adh null mutant as an internal standard. Several Slow and Fast allele pairs of different geographic origins were analyzed. The results provide strong evidence that the ADH protein concentration difference is not accounted for by RNA level.

Use of P-element-mediated transformation to identify the molecular basis of naturally occurring variants affecting Adh expression in Drosophila melanogaster.

The purpose of the work reported here is to identify the molecular basis of the difference in level of expression between the polymorphic Slow and Fast alcohol dehydrogenase (Adh) alleles in Drosophila melanogaster. Previous studies have shown that Fast lines typically have a two- to threefold higher activity level than Slow lines and they also have a substantially higher level of ADH-protein (estimated immunologically). The results of a restriction fragment length polymorphism study in relation to ADH activity variation had previously suggested that the difference in Adh expression between allozymes might not be due entirely to the amino acid replacement substitution, but could be due in part to linkage disequilibrium with a regulatory site polymorphism. Here we describe an approach that makes use of P-element-mediated transformation in order to identify the nucleotide substitution(s) responsible for this difference in ADH level. This approach consists of generating recombinants in vitro between Adh region clones derived from a typical Slow/Fast pair of alleles and then testing for the effects of particular restriction fragments on expression in vivo by transformation. Using this approach, the effect on both ADH activity and ADH-protein level clearly maps to a 2.3-kb restriction fragment that includes all of the Adh coding sequence and some intron and 3' flanking sequence, but excludes all of the 5' flanking sequence of the distal (adult) transcriptional unit. Comparison of Kreitman's DNA sequences for this fragment from several Slow and Fast alleles showing the typical difference in activity level shows that only three nucleotide substitutions distinguish all Fast from all Slow alleles. Thus, it is likely that one or more of these substitutions causes the major difference in Adh expression between allozymic classes. One of these substitutions is, of course, the Slow/Fast amino acid replacement substitution (at 1490) while the other two are nearby third position silent substitutions (at 1443 and 1527). A quantitative analysis of variation among transformant stocks shows that the P-element transformation approach can be used to localize even relatively small effects on gene expression (on the order of 20%).