Biology subjects

Clark, A. G.

Publications and source records attributed to Clark, A. G..

68 records · Page 4Linked to original sources

bioRxiv2018

Multiple loci of small effect confer wide variability in efficiency and resistance rate of CRISPR gene drive

Gene drives could allow for control of vector-borne diseases by directly suppressing vector populations or spreading genetic payloads designed to reduce pathogen transmission. CRISPR homing gene drives work by cleaving wild-type alleles, which are then converted to drive alleles by homology-directed repair, increasing the frequency of the drive in a population. However, resistance alleles can form when end-joining repair takes place in lieu of homology-directed repair. Such alleles cannot be converted to drive alleles, which would halt the spread of a drive through a population. To investigate the effects of natural genetic variation on resistance formation, we developed a CRISPR homing gene drive in Drosophila melanogaster and crossed it into the genetically diverse Drosophila Genetic Reference Panel (DGRP) lines, measuring several performance parameters. Most strikingly, resistance allele formation post-fertilization in the early embryo ranged from 7% to 79% among lines and averaged 42{+/-}18%. We performed a Genome-Wide Association Study (GWAS) using our results in the DGRP lines and found that the resistance and conversion rates were polygenic, with several genetic polymorphisms showing relatively weak association. RNAi knockdown of several of these genes confirmed their effect, but their small effect sizes implies that their manipulation will yield only modest improvements to the efficacy of gene drives.

genetics↗

bioRxiv2018

Molecular safeguarding of CRISPR gene drive experiments

CRISPR-based gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target sites and split drives as flexible safeguarding strategies for gene drive experiments.

genetics↗

bioRxiv2018

Allele frequency dynamics in a pedigreed natural population

A central goal of population genetics is to understand how genetic drift, natural selection, and gene flow shape allele frequencies through time. However, the actual processes underlying these changes - variation in individual survival, reproductive success, and movement - are often difficult to quantify. Fully understanding these processes requires the population pedigree, the set of relationships among all individuals in the population through time. Here, we use extensive pedigree and genomic information from a long-studied natural population of Florida Scrub-Jays (Aphelocoma coerulescens) to directly characterize the relative roles of different evolutionary processes in shaping patterns of genetic variation through time. We performed gene dropping simulations to estimate individual genetic contributions to the population and model drift on the known pedigree. We found that observed allele frequency changes are generally well predicted by accounting for the different genetic contributions of founders. Our results show that the genetic contribution of recent immigrants is substantial, with some large allele frequency shifts that otherwise may have been attributed to selection actually due to gene flow. We identified a few SNPs under directional short-term selection after appropriately accounting for gene flow. Using models that account for changes in population size, we partitioned the proportion of variance in allele frequency change through time. Observed allele frequency changes are primarily due to variation in survival and reproductive success, with gene flow making a smaller contribution. This study provides one of the most complete descriptions of short-term evolutionary change in allele frequencies in a natural population to date.

evolutionary biology↗

bioRxiv2018

Maximum likelihood estimation of fitness components in experimental evolution

Estimating fitness differences between allelic variants is a central goal of experimental evolution. Current methods for inferring selection from allele frequency time series typically assume that evolutionary dynamics at the locus of interest can be described by a fixed selection coefficient. However, fitness is an aggregate of several components including mating success, fecundity, and viability, and distinguishing between these components could be critical in many scenarios. Here we develop a flexible maximum likelihood framework that can disentangle different components of fitness and estimate them individually in males and females from genotype frequency data. As a proof-of-principle, we apply our method to experimentally-evolved cage populations of Drosophila melanogaster, in which we tracked the relative frequencies of a loss-of-function and wild-type allele of yellow. This X-linked gene produces a recessive yellow phenotype when disrupted and is involved in male courtship ability. We find that the fitness costs of the yellow phenotype take the form of substantially reduced mating preference of wild-type females for yellow males, together with a modest reduction in the viability of yellow males and females. Our framework should be generally applicable to situations where it is important to quantify fitness components of specific genetic variants, including quantitative characterization of the population dynamics of CRISPR gene drives.

evolutionary biology↗

bioRxiv2018

Sexual Conflict through Mother’s Curse and Father’s Curse

In contrast with autosomes, lineages of sex chromosomes reside for different amounts of time in males and females, and this transmission asymmetry makes them hotspots for sexual conflict. Similarly, the maternal inheritance of the mitochondrial genome (mtDNA) means that mutations that are beneficial in females can spread in a population even if they are deleterious in males, a form of sexual conflict known as Mothers Curse. While both Mothers Curse and sex chromosome induced sexual conflict have been well studied on their own, the interaction between mitochondrial genes and genes on sex chromosomes is poorly understood. Here, we use analytical models and computer simulations to perform a comprehensive examination of how transmission asymmetries of nuclear, mitochondrial, and sex chromosome-linked genes may both cause and resolve sexual conflicts. For example, the accumulation of male-biased Mothers Curse mtDNA mutations will lead to selection in males for compensatory nuclear modifier loci that alleviate the effect. We show how the Y chromosome, being strictly paternally transmitted provides a particularly safe harbor for such modifiers. This analytical framework also allows us to discover a novel kind of sexual conflict, by which Y chromosome-autosome epistasis may result in the spread of male beneficial but female deleterious mutations in a population. We christen this phenomenon Fathers Curse. Extending this analytical framework to ZW sex chromosome systems, where males are the heterogametic sex, we also show how W-autosome epistasis can lead to a novel kind of nuclear Mothers Curse. Overall, this study provides a comprehensive framework to understand how genetic transmission asymmetries may both cause and resolve sexual conflicts.

evolutionary biology↗

bioRxiv2018

Genus-wide sequencing supports a two-locus model for sex-determination in Phoenix

The date palm tree is a commercially important member of the genus Phoenix whose 14 species are all dioecious with separate male and female individuals. Previous studies identified a multi-megabase region of the date palm genome linked to sex and showed that dioecy likely developed in Phoenix prior to speciation. To identify genes critical to sex determination we sequenced the genomes of 28 Phoenix trees representing all 14 species. Male-specific sequences were identified and extended using phased single molecule sequencing or BAC clones to distinguish X and Y alleles.\n\nHere we show that only four genes contain sequences conserved in all analyzed males, likely identifying the changes foundational to dioecy in Phoenix. The majority of these sequences show similarity to a single genomic locus in the closely related oil palm. CYP703 and GPAT3, two genes critical to male flower development in other monocots, appear fully deleted in females while maintained as single copy in males. A LOG-like gene appears translocated into the Y chromosome and a cytidine deaminase-like appears at the border of a chromosomal rearrangement. Our data supports a two-mutation model for the evolution from hermaphroditism to dioecy through a gynodioecious intermediate.

genomics↗

bioRxiv2017

Genomic responses to selection for tame/aggressive behaviors in the silver fox (Vulpes vulpes)

Animal domestications have led to a shared spectrum of striking behavioral and morphological changes. To recapitulate this process, silver foxes have been selectively bred for tame and aggressive behaviors for over 50 generations at the Institute for Cytology and Genetics in Novosibirsk, Russia. To understand the genetic basis and molecular mechanisms underlying the phenotypic changes, we profiled gene expression level and coding SNP allele frequencies in two brain tissues from 12 aggressive and 12 tame foxes. Expression analysis revealed 146 genes in prefrontal cortex and 33 genes in basal forebrain that were differentially expressed (5% FDR). These candidates include genes in key pathways known to be critical to neurological processing, including the serotonin and glutamate receptor pathways. In addition, 295 of the 31,000 exonic SNPs show significant allele frequency differences between tame and aggressive population (1% FDR), including genes with a role in neural crest cell fate determination.

evolutionary biology↗

bioRxiv2017

Selection constrains high rates of satellite DNA mutation in Daphnia pulex

A long-standing evolutionary puzzle is that all eukaryotic genomes contain large amounts of tandemly-repeated satellite DNA whose composition varies greatly among even closely related species. To elucidate the evolutionary forces governing satellite dynamics, quantification of the rates and patterns of mutations in satellite DNA copy number and tests of its selective neutrality are necessary. Here we used whole-genome sequences of 28 mutation accumulation (MA) lines of Daphnia pulex in addition to six isolates from a non-MA population originating from the same progenitor to both estimate mutation rates of abundances of satellite sequences and evaluate the selective regime acting upon them. We found that mutation rates of individual satellite sequence \"kmers\" were both high and highly variable, ranging from additions/deletions of 0.29 - 105 copies per generation (reflecting changes of 0.12 - 0.80 percent per generation). Our results also provide evidence that new kmer sequences are often formed from existing ones. The non-MA population isolates showed a signal of either purifying or stabilizing selection, with 33 % lower variation in kmer abundance on average than the MA lines, although the level of selective constraint was not evenly distributed across all kmers. The changes between many pairs of kmers were correlated, and the pattern of correlations was significantly different between the MA lines and the non-MA population. Our study demonstrates that kmer sequences can experience extremely rapid evolution in abundance, which can lead to high levels of divergence in genome-wide satellite DNA composition between closely related species.

evolutionary biology↗

bioRxiv2017

Reducing resistance allele formation in CRISPR gene drives

CRISPR gene drives can efficiently convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. This mechanism could be used, for example, to rapidly disseminate a genetic payload through a population, promising novel strategies for the control of vector-borne diseases. However, all CRISPR gene drives tested have produced significant quantities of resistance alleles that cannot be converted to drive alleles and would likely prevent these drives from spreading in a natural population. In this study, we assessed three strategies for reducing resistance allele formation. First, we directly compared drives with the nanos and vasa promoters, which showed that the vasa drive produced high levels of resistance alleles in somatic cells. This was not observed in the nanos drive. Another strategy was the addition of a second gRNA to the drive, which both significantly increased the drive conversion efficiency and reduced the formation rate of resistance alleles. Finally, to minimize maternal carryover of Cas9, we assessed the performance of an autosomal drive acting in the male germline, and found no subsequent formation of resistance alleles in embryos. Our results mark a step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further reduction of resistance rates.

genetics↗

bioRxiv2017

Position Effect Variegation In Natural Populations Not Explained By Common Variation In Known Modifiers

Changes in chromatin state may drive changes in gene expression, and it is of growing interest to understand the population genetic forces that drive differences in chromatin state. Here, we use the phenomenon of position effect variegation (PEV), a well-studied proxy for chromatin state, to explore the genetic architecture of natural variation in factors that modify chromatin state. While previous mutation screens have identified over 150 suppressors and enhancers of PEV, it remains unknown to what extent allelic variation in these modifiers mediates inter-individual variation in chromatin state. Is natural variation in PEV mediated by segregating variation in known Su(var) and E(var) genes, or is the trait polygenic, with many variants mapping elsewhere in the genome? We designed a mapping study that directly answers this question and suggests that the bulk of the variance in PEV does not map to genes with prior annotated impact to PEV. Instead, we find enrichment of top P-value ranked associations that suggest impact to active promoter and transcription start site proximal regions. This work provides a quantitative view of the role naturally segregating autosomal variants play in modifying PEV, a phenomenon that continues to shape our understanding of chromatin state and epigenetics.

genetics↗

bioRxiv2017

Novel CRISPR/Cas9 gene drive constructs in Drosophila reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations

A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Though CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles. In this study, we developed two CRISPR gene drive constructs based on the nanos and vasa promoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organism Drosophila melanogaster. We observed resistance allele formation at high rates both prior to fertilization in the germline and post-fertilization in the embryo due to maternally deposited Cas9. Assessment of drive activity in genetically diverse backgrounds further revealed substantial differences in conversion efficiency and resistance rates. Our results demonstrate that the evolution of resistance will likely impose a severe limitation to the effectiveness of current CRISPR gene drive approaches, especially when applied to diverse natural populations.

genetics↗

bioRxiv2017

Evolutionary dynamics of male reproductive genes in the Drosophila virilis subgroup

Postcopulatory sexual selection (PCSS) is a potent evolutionary force that can drive rapid changes of reproductive genes within species, and thus has the potential to generate reproductive incompatibilities between species. Male seminal fluid proteins (SFPs) are major players in postmating interactions, and likely the main targets of PCSS in males. The virilis subgroup of Drosophila exhibits strong interspecific gametic incompatibilities, and can serve as a model to study the genetic basis of PCSS and gametic isolation. However, reproductive genes in this group have not been characterized. Here we use short-read RNA sequencing of male reproductive organs to examine the evolutionary dynamics of reproductive genes in members of the virilis subgroup: D. americana, D. lummei, D. novamexicana, and D. virilis. For each of the three male reproductive organs (accessory glands, ejaculatory bulb, and testes), we identify genes that show strong expression bias in a given tissue relative to the remaining tissues. We find that the majority of male reproductive transcripts are testes-biased, accounting for ~15% of all annotated genes. Ejaculatory bulb-biased transcripts largely code for lipid metabolic enzymes, and contain orthologs of the D. melanogaster ejaculatory bulb protein, Peb-me, which is involved in mating-plug formation. In addition, we identify 71 candidate SFPs, and show that this set of genes has the highest rate of nonsynonymous codon substitution relative to testes- and ejaculatory bulb-biased genes. Furthermore, these SFPs are underrepresented on the X chromosome and are enriched for proteolytic enzymes, which is consistent with SFPs in other insect species. Surprisingly, we find 35 D. melanogaster SFPs with conserved accessory gland expression in the virilis group, suggesting these genes may have conserved reproductive roles in Drosophila. Finally, we show that several of the SFPs that have the highest rate of nonsynonymous codon substitutions reside on the centromeric half of chromosome 2, which contributes to paternal gametic incompatibility between species. Our results suggest that SFPs are under strong selection in the virilis group, and likely play a major role in PCSS and/or gametic isolation.

evolutionary biology↗

bioRxiv2017

A pooled sequencing approach identifies a candidate meiotic driver in Drosophila

Meiotic drive occurs when a selfish element increases its transmission frequency above the Mendelian ratio by hijacking the asymmetric divisions of female meiosis. Meiotic drive causes genomic conflict and potentially has a major impact on genome evolution, but only a few drive loci of large effect have been described. New methods to reliably detect meiotic drive are therefore needed, particularly for discovering moderate-strength drivers that are likely to be more prevalent in natural populations than strong drivers. Here we report an efficient method that uses sequencing of large pools of backcross (BC1) progeny to test for deviations from Mendelian segregation genome-wide of single-nucleotide polymorphisms (SNPs) that distinguish the parental strains. We show that meiotic drive can be detected by a characteristic pattern of decay in distortion of SNP frequencies, caused by recombination unlinking the driver from distal loci. We further show that control crosses allow allele-frequency distortion caused by meiotic drive to be distinguished from distortion resulting from developmental effects. We used this approach to test whether chromosomes with extreme telomere-length differences segregate at Mendelian ratios, as telomeric regions are a potential hotspot for meiotic drive due to their roles in meiotic segregation and multiple observations of high rates of telomere sequence evolution. Using four different pairings of long and short telomere strains, we find no evidence that extreme telomere-length variation causes meiotic drive in Drosophila. However, we identify one candidate meiotic driver in a centromere-linked region that shows an ~8% increase in transmission frequency, corresponding to a ~54:46 segregation ratio. Our results show that candidate meiotic drivers of moderate strength can be readily detected and localized in pools of F1 progeny.

genetics↗

bioRxiv2016

Deconstructing isolation-by-distance: the genomic consequences of limited dispersal

Geographically limited dispersal can shape genetic population structure and result in a correlation between genetic and geographic distance, commonly called isolation-bydistance. Despite the prevalence of isolation-by-distance in nature, to date few studies have empirically demonstrated the processes that generate this pattern, largely because few populations have direct measures of individual dispersal and pedigree information. Intensive, long-term demographic studies and exhaustive genomic surveys in the Florida Scrub-Jay (Aphelocoma coerulescens) provide an excellent opportunity to investigate the influence of dispersal on genetic structure. Here, we used a panel of genome-wide SNPs and extensive pedigree information to explore the role of limited dispersal in shaping patterns of isolation-by-distance in both sexes, and at an exceedingly fine spatial scale (within ~10 km). Isolation-by-distance patterns were stronger in male-male and male-female comparisons than in female-female comparisons, consistent with observed differences in dispersal propensity between the sexes. Using the pedigree, we demonstrated how various genealogical relationships contribute to fine-scale isolation-by-distance. Simulations using field-observed distributions of male and female natal dispersal distances showed good agreement with the distribution of geographic distances between breeding individuals of different pedigree relationship classes. Furthermore, we extended Malecots theory of isolation-by-distance by building coalescent simulations parameterized by the observed dispersal curve, population density, and immigration rate, and showed how incorporating these extensions allows us to accurately reconstruct observed sex-specific isolation-by-distance patterns in autosomal and Z-linked SNPs. Therefore, patterns of fine-scale isolation-by-distance in the Florida Scrub-Jay can be well understood as a result of limited dispersal over contemporary timescales.\n\nAuthor SummaryDispersal is a fundamental component of the life history of most organisms and therefore influences many biological processes. Dispersal is particularly important in creating genetic structure on the landscape. We often observe a pattern of decreased genetic relatedness between individuals as geographic distances increases, or isolation-by-distance. This pattern is particularly pronounced in organisms with extremely short dispersal distances. Despite the ubiquity of isolation-by-distance patterns in nature, there are few examples that explicitly demonstrate how limited dispersal influences spatial genetic structure. Here we investigate the processes that result in spatial genetic structure using the Florida Scrub-Jay, a bird with extremely limited dispersal behavior and extensive genome-wide data. We take advantage of the long-term monitoring of a contiguous population of Florida Scrub-Jays, which has resulted in a detailed pedigree and measurements of dispersal for hundreds of individuals. We show how limited dispersal results in close genealogical relatives living closer together geographically, which generates a strong pattern of isolation-by-distance at an extremely small spatial scale (<10 km) in just a few generations. Given the detailed dispersal, pedigree, and genomic data, we can achieve a fairly complete understanding of how dispersal shapes patterns of genetic diversity over short spatial scales.

evolutionary biology↗