Browsing by Author "Michael D. Purugganan, Committee Member"

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Assessing Genetic Contributions to Performance of Communally Reared Families of Wild and Domesticated Reciprocal Hybrid Striped Bass.
(2006-07-18) Garber, Amber Frances; Craig V. Sullivan, Committee Chair; Ronald G. Hodson, Committee Member; Michael D. Purugganan, Committee Member; Bryon R. Sosinski, Committee Member
Expansion of the hybrid striped bass (HSB) aquaculture industry has been limited by high production costs dictating high prices. Much of the industry is dependant upon wild or captive broodstock. Selective breeding for an improved HSB may be one way to increase production efficiency driving down market prices and allowing industry expansion. Researchers at North Carolina State University have domesticated the HSB parental species over several generations (white bass, Morone chrysops and striped bass, M. saxatilis). This study compares performance (e.g., survival, growth) of HSB progeny from domesticated and wild broodstock that were communally reared in research and commercial ponds and provides an initial assessment of the degree to which important production traits have a genetic basis amenable to improvement. HSB progeny were produced using commercial in vitro spawning techniques and a nested mating design with multiple white bass dams crossed with individual striped bass sires. At the time of spawning, cross combinations were recorded and blood (DNA) samples were collected from the broodstock. These samples were used to test the variability of 68 microsatellite DNA markers. The seven most variable markers were multiplexed and used for progeny identification (whole larvae and fin clips). Initial progeny samples were collected and genotyped at 2 days post hatch (dph) from aquaria of mixed parentage (N = 580) revealing a large variation in initial survival (0-68.9%). Larval samples (N = 438) were also collected and genotyped at 3-4 dph (Phase 0) after domesticated and wild progeny were mixed (volumetric ratio of 48:52). Fin clip samples were collected from progeny at the end of Phases I (≥ 35 dph; N = 761), II (~1 year of age; N = 909) and III (~1.5 years of age, market; N = 2789). Domesticated progeny experienced an increased rate of mortality within the first ~35 dph. This may have been a result of several beginning factors (e.g., variations in female broodstock fitness and initial stocking times) as significant variations were mainly evident between Phase 0 and Phase I-III progeny samples (pairwise comparisons of allelic frequency distributions P ≤ 0.00909, chi-square analyses P < 0.0001), whereas comparisons between later Phases (I-III) were less likely to differ significantly. Large variations in family sizes prevented comparisons between progeny from all full sibling families (n = 33), but comparisons of progeny from paternal half sibling families (n = 5) was possible. Trends in ranked means of paternal half sibling progeny by length and weight indicated that progeny from domesticated crosses performed as well as or better than progeny from wild crosses. The progeny from domesticated crosses were a rounder shape by comparison of mean condition factors and landmark based geometric morphometric analysis (N = 1203 digital images in Phase III). The occurrence of external abnormalities differed between ponds as did the shape of HSB progeny (P = 0.0001) with a significant interaction effect for shape between ponds and paternal half sibling families (P = 0.0080) indicating large environmental effects. In Phase III progeny, females grew faster than males (P ≤ 0.0002) and also varied in shape (P < 0.0001), but an interaction between family and shape did not appear to exist. The full sibling families with the largest numbers of progeny were compared to determine differences in growth (length, weight) and shape. These comparisons indicated that an additive effect from both dams and sires exists in HSB progeny, however the study design did not allow for quantification of this effect. All of these results suggest that domesticated broodstock have maintained their value for commercial production of HSB and significant family variation is present that may be amenable to selective improvement.
Association mapping of major starch biosynthesis genes in Zea mays ssp. mays.
(2004-01-06) Wilson, Larissa Mary; Edward S. Buckler, IV, Committee Chair; Rebecca S. Boston, Committee Co-Chair; Michael D. Purugganan, Committee Member
Improving maize yield by utilizing natural allelic diversity is a major objective of today's breeders, and has likely been a goal since maize domestication. Starch is the main component of maize yield, and is an important agronomic trait needed for a wide range of uses from human and animal consumption to ethanol production. The level of starch in the maize kernel is controlled by upwards of 20 different loci, and has been the focus of multiple quantitative trait loci (QTL) studies in order to find regions in the maize genome that affect both starch content and starch quality, like the amylose/amylopectin ratio. The objective of this study was to evaluate the starch biosynthesis pathway using an association mapping approach, by evaluating six starch candidate genes from a diverse set of maize germplasm: Ae1, Bt2, Sh1, Sh2, Su1, and Wx1. The six starch candidate genes were amplified, sequenced, and aligned from 29 inbred lines and then evaluated for the level of diversity present. Estimates of π (nucleotide diversity) indicated, on average, starch genes contained 2.3- and 4.8-fold lower amounts of diversity at silent and nonsynonymous sites, respectively, than 20 randomly sampled genes from chromosome one of maize. Three of the starch loci (Ae1, Bt2, and Su1) had dramatic drops in diversity compared to Zea mays ssp. parviglumis. Furthermore, Hudson-Kreitman-Aguade (HKA) tests for selection were significant for these same three loci. In addition, another test for selection, Tajima?fs D, was significant at Ae1. These data suggest selection on starch genes has lowered diversity in the starch pathway. Smaller regions throughout each gene were sampled and aligned in a larger set of 97 maize inbreds for association tests. Phenotypic measurements of kernel composition (starch, protein, oil) and viscoamylographic (viscosity, pasting) profiles of starch were used in separate principle component analyses for the association tests. Significant associations (P< 0.05) with kernel composition traits, while controlling for population structure, were found in Sh1, Sh2, and Bt2. Significant associations for starch pasting traits were found in Sh1, Sh2, and Ae1. Possible phenotypic effects were examined between alleles with significant associations. For kernel composition traits, Sh1and Sh2 showed a general genotype by environment (G X E) effect. In Bt2, a nonsynonymous change at residue 22 caused lower variance in oil content. For starch pasting traits, an allele in Sh1 caused a 1% increase in pasting temperature. At Ae1, a nonsynonymous change at residue 58 had a 1.6% higher pasting temperature and 4.6% higher amylose content. A nonsynonymous change at residue 318 in Sh2 caused a 6% increase in amylose. This study supports previous findings that the Sh2 locus affects amylose content, but has offered much higher resolution than is possible with traditional linkage mapping, while examining a much broader range of alleles. Therefore, even in a moderately heritable pathway, such as the starch biosynthesis pathway, association methods can be successful in narrowing down regions of effect, most times within 1000 bp.
Disease Gene Mapping in General Pedigrees
(2005-02-28) Li, Li; Michael D. Purugganan, Committee Member; Bruce S. Weir, Committee Chair; Sharon R. Browning, Committee Member; Zhao-Bang Zeng, Committee Member; Margarate G. Ehm, Committee Member
Disease gene mapping is one of the main focuses of genetic epidemiology and statistical genetics. This dissertation explores some methods and algorithms in this area, especially in pedigrees. The first chapter gives an introduction to human genetics and disease gene mapping. Existing linkage and association methods are introduced and compared. Probabilities of genotypic data from multiple linked marker loci on related individuals are used as likelihoods of gene locations for gene-mapping, or as likelihoods of other parameters of interest in human genetics. With the recent development in genetics and molecular biology techniques, large-scale marker data has become available, which requires highly efficient likelihood calculations especially for complex pedigrees. Algorithms for likelihood calculations for pedigree data are reviewed in chapter 2. Besides exact likelihood calculation methods and MCMC, a Sequential Importance Sampling (SIS) approach has been proposed to enable calculations for large pedigrees with large numbers of markers. However, when the system gets large, the variance of the importance sampling weights increases while both efficiency and accuracy of the method decrease. We propose an optimization algorithm for calculating the likelihood of general pedigrees in Chapter 3. We incorporate a resampling strategy into SIS to reduce the variance inflation problem. A successful linkage analysis may identify a linkage region of interest containing hundreds of genes at a magnitude of perhaps ten to thirty centiMorgans. A follow-up association (or so-called linkage disequilibrium) analysis can provide much finer gene-mapping but is subject to greater multiple testing problems. In Chapter 4, we present a method for determining whether an association result is responsible for a non-parametric linkage result for binary traits in general pedigrees. The correlation between family frequency of a variant of interest and family LOD score is used as a measure of whether the association between a given variant at a marker and the disease status can help to explain a significant linkage result seen in the collection of families in the region around the marker.
Estimation and Sampling Properties of Gene Diversity, Heterozygosity and F[subscript ST]
(2004-12-30) Johnson, Andrea Michelle; Bruce S. Weir, Committee Co-Chair; William R. Atchley, Committee Co-Chair; Jeffrey L. Thorne, Committee Member; Michael D. Purugganan, Committee Member
Estimates of the coancestry coefficient F[subscript ST], gene diversity and heterozygosity have been used in many fields, including conservation and evolutionary biology, and forensic studies. Although the sampling properties of estimators of these parameters could affect inferences to be made, these continue to be frequently overlooked in published analyses. This dissertation characterizes the estimators of these measures by presenting relevant theoretical developments, approaches to estimation, and results regarding evaluations of the sampling properties of these three measures. Making inferences about the genetic variation among populations of a species, rather than some larger, between-species scope, will be the biological focus. The accuracy and precision of the method of moments and maximum likelihood estimators of population-specific F[subscript ST] developed by citet[Weir02] are evaluated through population simulation and analysis of an empirical data set. Of the two estimators considered, the method of moments estimator for population-specific F[subscript ST] is found to be relatively unbiased with a large sampling variance, which increases as coancestry increases in a population. Sampling more loci has a much stronger effect on reducing this sampling variance than sampling more individuals. The other estimator evalutated here obtained by maximum-likelihood poorly estimates the coancestry in a population for two iterative approaches and a non-iterative approach, and is not recommended for future analyses. Problems with estimates obtained from individual loci with very low polymorphism levels for both estimators are discussed and practical measures for proceeding with analyses are suggested. Properties of several methods for inferring the variances of sample heterozygosity or gene diversity are evaluated, including the use of a new random model for the total variance of sample heterozygosity. Large differences with a previous mixed model are observed for a case where there is a large variance component due to loci. Several approximations are evaluated and compared to variances obtained from exact expressions. Different results with unbalanced data for the total variance of sample heterozygosity are obtained with four variance component methods, as expected by statistical theory. The likelihood-based methods considered here are shown to be robust to violations of assumptions of normality, even for very small sample sizes.
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.
Molecular and evolutionary analysis of the GATA transcription factor family
(2003-03-03) Lowry, Jason Allen; William R. Atchley, Committee Chair; Steven L. Spiker, Committee Member; Michael D. Purugganan, Committee Member; Jeffrey L. Thorne, Committee Member
The objective of this research has been to characterize the evolution of the GATA family of transcription factors through phylogenetic, molecular, and biochemical analyses. From a phylogenetic perspective, we address three major questions. First, does this protein family represent a monophyletic or polyphyletic group? Second, what methods of gene or modular duplication are utilized within different organisms to propagate and maintain this group of proteins? Third, what are the structural or functional constraints on evolution of the conserved DNA-binding domain? These questions are addressed through a combination of computational and molecular methods. Phylogenetic analyses provide evidence of monophyletic origin for the GATA family followed by gene duplication and modular evolution, accompanied by considerable divergence outside the conserved zinc finger DNA-binding domain. Genomic comparisons permit the tracing of GATA factor evolution and provide insight into mechanisms utilized by respective organisms. Finally, mutational and biochemical analyses enable the separation of phylogenetic and structural/functional constraints on the conserved zinc finger DNA-binding domain. The result of this research is a predictive motif for classifying potentially homologous proteins to be discovered in future studies.
Molecular Evolution and Population Genetics of Tomato spotted wilt virus (TSWV).
(2005-12-10) Tsompana, Maria; George G. Kennedy, Committee Member; David F. Ritchie, Committee Member; Michael D. Purugganan, Committee Member; J W Moyer, Committee Chair
The overall goal of this dissertation research was to elucidate the molecular evolution and population genetics of Tomato Spotted Wilt virus (TSWV), at the species level and within individual isolates, and to develop a standardized diagnostic system that can be used to assign attribution to initial TSWV infections. Initially, using consensus sequence data from genes encoding five viral proteins we applied a multilocus molecular population genetic framework to characterize the genetic status and recent evolutionary history of the TSWV species. Our analysis provided the first demonstration of population structuring and species-wide population expansions for TSWV, attributed possibly to founder effects. Also, we identified positive selection favoring divergence between Tospovirus species and purifying selection acting at the species level to preserve protein function. In addition, we were able to discover specific amino acid sites subject to positive selection within Bunyaviridae and to estimate the level of genetic heterogeneity of the TSWV species. Subsequently, in order to characterize the population history and genetic structure of individual wild-type TSWV isolates, thirteen geographically and host-diverse isolates were amplified, cloned and 516 clones were sequenced. Estimation of levels of genetic diversity and haplotype analysis revealed that natural TSWV isolates are highly heterogeneous viral populations that consist of one or more haplotypes with high frequency and an array of closely related rare haplotypes, some of which are defective. These viral populations exhibit a high transitional bias, attributed to the function of RNA-dependent RNA polymerase or an editing enzyme such as dsRAD. Also high levels of among-population differentiation were observed induced by geographic and/or host related factors. Demographic analysis based on tests of neutrality, gene genealogies and the coalescent revealed an excess of rare polymorphism and a shallow population genetic architecture consistent with a model of population growth for all analyzed TSWV isolates. Finally, data from genes encoding two viral proteins (NSm and L) were used for analysis of optimal informational content and for phylogenetic analysis. Our research has identified partial sequence regions that contain similar phylogenetic information and perform as well as the complete NSm and RdRp genes, for branching points statistically supported (bootstrap value>50%). We propose a new advanced diagnostic system, which will use the NSm and RdRp local regions together with the N gene of TSWV to assign attribution to initial TSWV infections and prevent their spread to an epidemic form.
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.