Browsing by Author "Trudy F. C. Mackay, Committee Chair"

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The Genetic Architecture of Complex Traits: Starvation Resistance in Drosophila melanogaster
(2004-09-02) Harbison, Susan Tracy; Trudy F. C. Mackay, Committee Chair; Michael D. Purugganan, Committee Member; Gregory C. Gibson, Committee Member; Bruce S. Weir, Committee Member
In nature, animals are often subjected to periods of sub-optimal food resources. Characteristic responses to starvation stress have been observed in bacteria, nematodes and yeast: they alter their morphology, become quiescent, and suspend reproduction until adequate food resources become available. Studies of laboratory and natural populations of Drosophila reveal a surprising amount of genetic variation for starvation tolerance. The presence of this genetic variation is an evolutionary puzzle, as variability would not be expected in a key trait related to individual survival. While starvation resistance has been positively correlated with lifespan and other stresses, it is often negatively correlated with fecundity, suggesting that a trade-off between reproduction and individual survival might be present. In order to evaluate this hypothesis, the suite of genes affecting starvation resistance and their properties must be known. Three complementary methods were used to identify genes affecting starvation resistance: a P-element insertional mutagenesis screen, which directly identifies candidate genes involved in the response to starvation stress; deficiency complementation mapping, which reveals small genomic regions contributing to variation in starvation resistance; and transcriptome analysis using microarrays, which has the potential to identify both types of genes. The starvation tolerance phenotype was assessed for 933 P-element insertion lines in two isogenic backgrounds: Canton-S and Samarkand. 383 insertions had a significant effect on starvation tolerance. The effect of the P-element inserts was generally negative and often sex-specific. Only 31 insertions significantly increased starvation tolerance. Significant insertions tag genes that are putatively involved in the starvation stress response. Deficiency complementation mapping was used to fine-map broad genomic regions (quantitative trait loci, or QTL) previously identified for starvation resistance. The five original QTL fractionated into thirteen smaller QTL, six of which had sex-specific effects. From these fine-mapped regions 26 genes were chosen for mutation complementation testing. Twelve of the 26 genes showed a significant effect on variation in starvation resistance between the two wild-type strains, Oregon-R and 2b. Transcriptome analysis was performed on a subset of the recombinant inbred mapping population used to identify broad QTL affecting starvation resistance: two lines resistant to starvation, two lines susceptible to starvation, and the two parental lines, Oregon-R and 2b. RNA samples were obtained in both the unstarved and starved states for these lines. Many genes were involved in the response to starvation stress: 3,528 unique probe sets exhibited significant differences in transcript abundance between the unstarved and starved states. 217 probe sets were identified as QTL that may affect variation in starvation resistance. 47 of these probe sets fell within the original QTL regions; nine probe sets fell within fine-mapped QTL found from the deficiency complementation tests. Further analysis revealed substantial epistasis at both the transcript level and the level of the phenotype, adding unexpected complexity to the attempt to map QTL using microarrays. Many of the genes implicated in this study have known phenotypes in cell fate specification/proliferation, feeding behavior, oogenesis, and metabolism, suggesting extensive pleiotropy. Mutational and transcriptional effects were often sex-specific. The large numbers of genes identified in this study suggest that a balance between mutation and selection may maintain variation in starvation resistance, rather than a trade-off among life history traits.
The Quantitative Genetic Basis of Mating Behavior and Speciation in Drosophila.
(2004-01-12) Moehring, Amanda Jean; Trudy F. C. Mackay, Committee Chair; Gregory C. Gibson, Committee Co-Chair; Coby Schal, Committee Member; Robert R. H. Anholt, Committee Member
The widely-accepted Biological Species Concept defines species as populations that are reproductively isolated, i.e. are unable to mate with one another or produce viable and fertile progeny when given the opportunity. Speciation is characterized by the evolution of prezygotic (ethological barriers to interspecific mating) and postzygotic (reduced viability and fertility of interspecific hybrids) reproductive isolating mechanisms. Although recent progress has been made towards understanding the genetic basis of postzygotic isolation, little is known of the genetic architecture of sexual isolation — arguably the most important form of reproductive isolation in animals. In addition, it has not been determined if reproductive isolation occurs due to selection acting on variation within a species or arises from novel mutations. In order to understand how new species arise, the genetic basis of variation in mating behavior within a species, as well as the genetic basis for prezygotic reproductive isolation between species, must be known. The mating behavior of Drosophila consists of a series of actions that exchange auditory, visual and chemosensory signals between males and females. Although mating behavior has been studied extensively in Drosophila, most known genes affecting mating behavior have been located through the mutation of single genes. The wide range of variation in courtship behavior in natural populations is believed to arise from the joint segregation of multiple quantitative trait loci (QTL) with varying effects that can be influenced by the environment. Here, we identified QTL that affect courtship occurrence, courtship latency, copulation occurrence and copulation latency that segregate between a D. melanogaster strain selected for reduced male mating propensity (2b) and a standard wild-type strain (Oregon-R). Mating behavior was assessed in a population of 98 recombinant inbred lines derived from these two strains and QTL affecting mating behavior were mapped using composite interval mapping. There were four QTL affecting male mating behavior at cytological locations 1A;3E, 57C;57F, 72A;85F and 96F;99A. We used deficiency complementation mapping to map the autosomal QTL with much higher resolution to five QTL at 56F5;56F8, 56F9;57A2, 70E1;71F4, 78C5;79A1, and 96F1;97B1. Quantitative complementation tests performed for 45 positional candidate genes within these intervals revealed seven genes which failed to complement the QTL: eagle, 18 wheeler, Enhancer of split, Polycomb, spermatocyte arrest, l(2)05510 and l(2)k02206. None of these genes have been previously implicated in mating behavior, demonstrating that quantitative analysis of subtle variants can reveal novel pleiotropic effects of key developmental loci on behavior. In a separate experiment, we mapped QTL contributing to prezygotic reproductive isolation between Drosophila simulans and D. mauritiana. We mapped at least seven QTL affecting discrimination of D. mauritiana females against D. simulans males, three QTL affecting D. simulans male traits against which D. mauritiana females discriminate, and six QTL affecting D. mauritiana male traits against which D. simulans females discriminate. QTL affecting sexual isolation are largely different in males and females and between the two species, and are not preferentially located on the X chromosome. Relatively few QTL with moderate to large effects associated with pre-zygotic isolation facilitates future positional cloning of the underlying genes. In contrast to results for postzygotic isolation, no epistasis was detected between QTL for prezygotic isolation. Several of the intraspecific D. melanogaster mating behavior QTL overlap those found to affect reproductive isolation between D. simulans and D. mauritiana. Future testing of these positional candidate genes for their effect on reproductive isolation could provide evidence that speciation arises in response to selection acting on naturally-occurring variation in a population.
Quantitative Genetics and Genomics of Drosophila Life Span
(2006-11-10) Wilson, Rhonda Henderson; William R. Atchley, Committee Member; Gregory C. Gibson, Committee Member; Michael D. Purugganan, Committee Member; Trudy F. C. Mackay, Committee Chair
Limited life span and senescence are near-universal characteristics of eukaryotic organisms, controlled by many interacting quantitative trait loci (QTLs) with individually small effects, whose expression is sensitive to the environment. Understanding how genetic and environmental factors interact to limit life span and generate variation between individuals, populations and species, is important from both a human health and an evolutionary theory perspective. To begin to dissect the complex genetic architecture of longevity, it is necessary to identify the genes affecting life span and natural variation in life span. Here we have used quantitative complementation mapping to deficiencies, gene expression analysis, and functional tests to mutations at positional candidate genes to gain a better understanding of genes and categories of genes associated with the aging process. These complementary approaches have allowed us to identify several genomic regions as well as specific candidate genes affecting longevity and variation in longevity. Quantitative complementation tests to 69 overlapping deficiencies covering approximately 80% of the third chromosome yielded 11 QTLs affecting variation in life span between five old ("O") lines selected for postponed senescence and their five base ("B") control lines. Most QTLs were sex-specific, and all but one affected multiple O lines, suggesting that variation in life span for the B and selected O populations is most often attributable to the effects of common alleles. However, these 11 QTLs spanned over 4874 kb and contained approximately 598 genes. To identify and prioritize individual genetic loci affecting life span and variation in life span within our chromosomal regions as well as the remaining genome, we used whole genome expression analyses over multiple ages for one B and two O lines. Two separate analyses were used to compare changes in transcript abundance at the same chronological and physiological age between ages and lines. Over 26% of the genome was significantly altered between young and old flies and more than 5% of the genome showed significant changes between control and selected lines (indicating variation in aging effects) at multiple ages. Significant probe sets fell into a diverse group of biological processes and molecular functions, many associated with processes and pathways known to affect aging as well as many correlated traits in O lines. Examination of expression patterns for specific genes showed that O lines commonly exhibited a delayed response to aging, although different patterns of expression were observed as well. Transcriptional analyses were followed up with functional tests to mutants at positional candidate genes, which were selected, based on significant probe sets for either age or line effects and the availability of mutants. P-element insertion lines and their co-isogenic controls allowed us to test for age effects. Forty-four percent of 27 P-element mutants tested showed significant differences in mean life span from their co-isogenic control lines, with all but one decreasing life span. Quantitative complementation tests to these mutants provided an efficient method to test for variation in aging as 70% of the ten mutants tested yielded significant results. Candidate genes implicated in functional tests for both aging and variation in aging are involved in various categories of biological processes, including oogenesis, chromatin silencing, spermatogenesis, development, defense response, locomotor behavior, and cell death, suggesting that many of the processes that affect aging may affect variation in aging as well.
Quantitative Molecular Genetics of Longevity in Drosophila melanogaster.
(2004-08-18) Thornsberry, Gretchen Lindsay Geiger; Bruce Weir, Committee Member; Trudy F. C. Mackay, Committee Chair; Greg Gibson, Committee Member; Michael Purugganan, Committee Member
Limited life span and senescence are universal phenomena, controlled by genetic and environmental factors whose interactions both limit life span and generate variation in life span between individuals, populations and species. To understand the genetic architecture of aging it is necessary to know what loci affect variation in life span, what are the allelic effects at these loci and what molecular polymorphisms define quantitative trait locus (QTL) alleles. Here, quantitative complementation tests were used to determine whether candidate life span genes such as Superoxide dismutase (Sod), Catalase (Cat), heat shock proteins, DNA repair enzymes, glucose metabolism or male accessory gland proteins interact genetically with naturally occurring QTL affecting variation in life span in Drosophila melanogaster. Inbred strains derived from a natural population were crossed to stocks containing null mutations or deficiencies uncovering the above genes. Life span of the heterozygous progeny was assayed. A significant cross (mutant versus wild-type allele of the candidate gene) by inbred line interaction term from analysis of variance of the life span data indicates a genetic interaction between the candidate gene allele and the naturally occurring life span QTL. Of the sixteen candidate regions and genes tested, Df(2L)cl7, Df(3L)Ly, Df(3L)AC1, Df(3R)e-BS2, and α-Glycerol phosphate dehydrogenase showed significant failure to complement wild-type alleles in both sexes, and an Alcohol dehydrogenase mutant failed to complement in females. Several genes known to regulate life span (Sod, Cat, and rosy) complemented the life span effects of alleles, suggesting little natural variation affecting longevity at these loci, at least in this sample of alleles. Quantitative complementation tests are therefore useful for identifying candidate genes contributing to segregating genetic variation in life span in nature. Mutations in most vital genes can potentially affect life history traits, but it is not known what subset of these loci harbor naturally occurring variation affecting the rate of aging and the ability to resist stress. While the gene Punch (Pu) was not significant in the quantitative complementation test, it has been implicated in starvation resistance. As there is a direct relationship between stress resistance and longevity, Pu, which encodes GTP cyclohydrolase (GTPCH), is a candidate gene for associating molecular variation and variation in life pan. GTPCH regulates the catecholamine biosynthesis pathway by catalyzing the formation of tetrahydrobiopterin, the rate-limiting molecule, and by regulating tyrosine hydroxylase, a key enzyme in the pathway. The extent to which molecular variation at Pu contributes to phenotypic variation was assessed by associating single nucleotide polymorphisms (SNPs) at Pu with longevity. Nucleotide variation was determined for ten Pu alleles. Genotypes of 28 SNPs were determined on a sample of 178 isogenic second chromosomes sampled from the Raleigh, USA population and substituted into the highly inbred Samarkand background. Life span was determined for the chromosome substitution lines and the association between longevity phenotype and SNP genotype was assessed for each polymorphic marker. Three SNPs were significantly associated with life span (C6291A, P = 0.0183; A6389T, P = 0.0466; G6894C, P = 0.0024). None of these SNPs was significant individually following a permutation test accounting for multiple tests and partially correlated markers. However, the three SNPs associated with life span were in global linkage disequilibrium. Haplotypes of these SNPs were highly significantly associated with variation in longevity (P < 0.0001), and accounted for 13.5 % of the genetic variance and 1.86 % of the phenotypic variance in longevity attributable to chromosomes 2. As Pu is a regulator of the catecholamine biosynthetic pathway, these findings suggest the importance of the production of biogenic amines in determining variation for longevity.