Showing papers in "Genetics in 2021"

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans.

Abstract: The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

Assessing the Performance of qpAdm: A Statistical Tool for Studying Population Admixture

Abstract: qpAdm is a statistical tool for studying the ancestry of populations with histories that involve admixture between two or more source populations. Using qpAdm, it is possible to identify plausible models of admixture that fit the population history of a group of interest and to calculate the relative proportion of ancestry that can be ascribed to each source population in the model. Although qpAdm is widely used in studies of population history of human (and nonhuman) groups, relatively little has been done to assess its performance. We performed a simulation study to assess the behavior of qpAdm under various scenarios in order to identify areas of potential weakness and establish recommended best practices for use. We find that qpAdm is a robust tool that yields accurate results in many cases, including when data coverage is low, there are high rates of missing data or ancient DNA damage, or when diploid calls cannot be made. However, we caution against co-analyzing ancient and present-day data, the inclusion of an extremely large number of reference populations in a single model, and analyzing population histories involving extended periods of gene flow. We provide a user guide suggesting best practices for the use of qpAdm.

Rapid detection of inter-clade recombination in SARS-CoV-2 with Bolotie.

Abstract: The ability to detect recombination in pathogen genomes is crucial to the accuracy of phylogenetic analysis and consequently to forecasting the spread of infectious diseases and to developing therapeutics and public health policies. However, in case of the SARS-CoV-2, the low divergence of near-identical genomes sequenced over a short period of time makes conventional analysis infeasible. Using a novel method, we identified 225 anomalous SARS-CoV-2 genomes of likely recombinant origins out of the first 87,695 genomes to be released, several of which have persisted in the population. Bolotie is specifically designed to perform a rapid search for inter-clade recombination events over extremely large datasets, facilitating analysis of novel isolates in seconds. In cases where raw sequencing data was available, we were able to rule out the possibility that these samples represented co-infections by analyzing the underlying sequence reads. The Bolotie software and other data from our study are available at https://github.com/salzberg-lab/bolotie.

Phylogenomic approaches to detecting and characterizing introgression.

Abstract: Phylogenomics has revealed the remarkable frequency with which introgression occurs across the tree of life. These discoveries have been enabled by the rapid growth of methods designed to detect and characterize introgression from whole-genome sequencing data. A large class of phylogenomic methods makes use of data across species to infer and characterize introgression based on expectations from the multispecies coalescent. These methods range from simple tests, such as the D-statistic, to model-based approaches for inferring phylogenetic networks. Here, we provide a detailed overview of the various signals that different modes of introgression are expected leave in the genome, and how current methods are designed to detect them. We discuss the strengths and pitfalls of these approaches and identify areas for future development, highlighting the different signals of introgression, and the power of each method to detect them. We conclude with a discussion of current challenges in inferring introgression and how they could potentially be addressed.

Chemosensory signal transduction in Caenorhabditis elegans.

Abstract: Chemosensory neurons translate perception of external chemical cues, including odorants, tastants, and pheromones, into information that drives attraction or avoidance motor programs. In the laboratory, robust behavioral assays, coupled with powerful genetic, molecular and optical tools, have made Caenorhabditis elegans an ideal experimental system in which to dissect the contributions of individual genes and neurons to ethologically relevant chemosensory behaviors. Here, we review current knowledge of the neurons, signal transduction molecules and regulatory mechanisms that underlie the response of C. elegans to chemicals, including pheromones. The majority of identified molecules and pathways share remarkable homology with sensory mechanisms in other organisms. With the development of new tools and technologies, we anticipate that continued study of chemosensory signal transduction and processing in C. elegans will yield additional new insights into the mechanisms by which this animal is able to detect and discriminate among thousands of chemical cues with a limited sensory neuron repertoire.

Clade-specific chromosomal rearrangements and loss of subtelomeric adhesins in Candida auris.

Abstract: Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.

Regulation of growth and cell fate during tissue regeneration by the two SWI/SNF chromatin-remodeling complexes of Drosophila.

Abstract: To regenerate, damaged tissue must heal the wound, regrow to the proper size, replace the correct cell types, and return to the normal gene-expression program However, the mechanisms that temporally and spatially control the activation or repression of important genes during regeneration are not fully understood To determine the role that chromatin modifiers play in regulating gene expression after tissue damage, we induced ablation in Drosophila melanogaster imaginal wing discs, and screened for chromatin regulators that are required for epithelial tissue regeneration Here, we show that many of these genes are indeed important for promoting or constraining regeneration Specifically, the two SWI/SNF chromatin-remodeling complexes play distinct roles in regulating different aspects of regeneration The PBAP complex regulates regenerative growth and developmental timing, and is required for the expression of JNK signaling targets and the growth promoter Myc By contrast, the BAP complex ensures correct patterning and cell fate by stabilizing the expression of the posterior gene engrailed Thus, both SWI/SNF complexes are essential for proper gene expression during tissue regeneration, but they play distinct roles in regulating growth and cell fate

Drosophila sensory receptors-a set of molecular Swiss Army Knives.

Abstract: Genetic approaches in the fruit fly, Drosophila melanogaster, have led to a major triumph in the field of sensory biology-the discovery of multiple large families of sensory receptors and channels Some of these families, such as transient receptor potential channels, are conserved from animals ranging from worms to humans, while others, such as "gustatory receptors," "olfactory receptors," and "ionotropic receptors," are restricted to invertebrates Prior to the identification of sensory receptors in flies, it was widely assumed that these proteins function in just one modality such as vision, smell, taste, hearing, and somatosensation, which includes thermosensation, light, and noxious mechanical touch By employing a vast combination of genetic, behavioral, electrophysiological, and other approaches in flies, a major concept to emerge is that many sensory receptors are multitaskers The earliest example of this idea was the discovery that individual transient receptor potential channels function in multiple senses It is now clear that multitasking is exhibited by other large receptor families including gustatory receptors, ionotropic receptors, epithelial Na+ channels (also referred to as Pickpockets), and even opsins, which were formerly thought to function exclusively as light sensors Genetic characterizations of these Drosophila receptors and the neurons that express them also reveal the mechanisms through which flies can accurately differentiate between different stimuli even when they activate the same receptor, as well as mechanisms of adaptation, amplification, and sensory integration The insights gleaned from studies in flies have been highly influential in directing investigations in many other animal models

The fitness costs and benefits of trisomy of each Candida albicans chromosome.

Abstract: Candida albicans is a prevalent human fungal pathogen Rapid genomic change, due to aneuploidy, is a common mechanism that facilitates survival from multiple types of stresses including the few classes of available antifungal drugs The stress survival of aneuploids occurs despite the fitness costs attributed to most aneuploids growing under idealized lab conditions Systematic study of the aneuploid state in C albicans has been hindered by the lack of a comprehensive collection of aneuploid strains Here, we describe a collection of diploid C albicans aneuploid strains, each carrying one extra copy of each chromosome, all from the same genetic background We tested the fitness of this collection under several physiological conditions including shifts in pH, low glucose, oxidative stress, temperature, high osmolarity, membrane stress, and cell wall stress We found that most aneuploids, under most conditions, were less fit than their euploid parent, yet there were specific conditions under which specific aneuploid isolates provided a fitness benefit relative to the euploid parent strain Importantly, this fitness benefit was attributable to the change in the copy number of specific chromosomes Thus, C albicans can tolerate aneuploidy of each chromosome and some aneuploids confer improved growth under conditions that the yeast encounters in its host niches

The impacts of cytoplasmic incompatibility factor (cifA and cifB) genetic variation on phenotypes.

Abstract: Wolbachia are maternally transmitted, intracellular bacteria that can often selfishly spread through arthropod populations via cytoplasmic incompatibility (CI). CI manifests as embryonic death when males expressing prophage WO genes cifA and cifB mate with uninfected females or females harboring an incompatible Wolbachia strain. Females with a compatible cifA-expressing strain rescue CI. Thus, cif-mediated CI confers a relative fitness advantage to females transmitting Wolbachia. However, whether cif sequence variation underpins incompatibilities between Wolbachia strains and variation in CI penetrance remains unknown. Here, we engineer Drosophila melanogaster to transgenically express cognate and non-cognate cif homologs and assess their CI and rescue capability. Cognate expression revealed that cifA;B native to D. melanogaster causes strong CI, and cognate cifA;B homologs from two other Drosophila-associated Wolbachia cause weak transgenic CI, including the first demonstration of phylogenetic type 2 cifA;B CI. Intriguingly, non-cognate expression of cifA and cifB alleles from different strains revealed that cifA homologs generally contribute to strong transgenic CI and interchangeable rescue despite their evolutionary divergence, and cifB genetic divergence contributes to weak or no transgenic CI. Finally, we find that a type 1 cifA can rescue CI caused by a genetically divergent type 2 cifA;B in a manner consistent with unidirectional incompatibility. By genetically dissecting individual CI functions for type 1 and 2 cifA and cifB, this work illuminates new relationships between cif genotype and CI phenotype. We discuss the relevance of these findings to CI’s genetic basis, phenotypic variation patterns, and mechanism.

Genetic evaluation including intermediate omics features.

Abstract: In animal and plant breeding and genetics, there has been an increasing interest in intermediate omics traits, such as metabolomics and transcriptomics, which mediate the effect of genetics on the phenotype of interest. For inclusion of such intermediate traits into a genetic evaluation system, there is a need for a statistical model that integrates phenotypes, genotypes, pedigree, and omics traits, and a need for associated computational methods that provide estimated breeding values. In this paper, a joint model for phenotypes and omics data is presented, and a formula for the breeding values on individuals is derived. For complete omics data, three equivalent methods for best linear unbiased prediction of breeding values are presented. In all three cases, this requires solving two mixed model equation systems. Estimation of parameters using restricted maximum likelihood is also presented. For incomplete omics data, extensions of two of these methods are presented, where in both cases, the extension consists of extending an omics-related similarity matrix to incorporate individuals without omics data. The methods are illustrated using a simulated data set.

Heritability of DNA methylation in threespine stickleback (Gasterosteus aculeatus)

Abstract: Epigenetic mechanisms underlying phenotypic change are hypothesized to contribute to population persistence and adaptation in the face of environmental change To date, few studies have explored the heritability of intergenerationally stable methylation levels in natural populations, and little is known about the relative contribution of cis- and trans-regulatory changes to methylation variation Here, we explore the heritability of DNA methylation, and conduct methylation quantitative trait loci (meQTLs) analysis to investigate the genetic architecture underlying methylation variation between marine and freshwater ecotypes of threespine stickleback (Gasterosteus aculeatus) We quantitatively measured genome-wide DNA methylation in fin tissue using reduced representation bisulfite sequencing of F1 and F2 crosses, and their marine and freshwater source populations We identified cytosines (CpG sites) that exhibited stable methylation levels across generations We found that additive genetic variance explained an average of 24-35% of the methylation variance, with a number of CpG sites possibly autonomous from genetic control We also detected both cis- and trans-meQTLs, with only trans-meQTLs overlapping with previously identified genomic regions of high differentiation between marine and freshwater ecotypes Finally, we identified the genetic architecture underlying two key CpG sites that were differentially methylated between ecotypes These findings demonstrate a potential role for DNA methylation in facilitating adaptation to divergent environments and improve our understanding of the heritable basis of population epigenomic variation

Specific collagens maintain the cuticle permeability barrier in Caenorhabditis elegans.

Abstract: Collagen-enriched cuticle forms the outermost layer of skin in nematode Caenorhabditis elegans The nematode's genome encodes 177 collagens, but little is known about their role in maintaining the structure or barrier function of the cuticle In this study, we found six permeability determining (PD) collagens Loss of any of these PD collagens-DPY-2, DPY-3, DPY-7, DPY-8, DPY-9, and DPY-10-led to enhanced susceptibility of nematodes to paraquat (PQ) and antihelminthic drugs- levamisole and ivermectin Upon exposure to PQ, PD collagen mutants accumulated more PQ and incurred more damage and death despite the robust activation of antioxidant machinery We find that BLMP-1, a zinc finger transcription factor, maintains the barrier function of the cuticle by regulating the expression of PD collagens We show that the permeability barrier maintained by PD collagens acts in parallel to FOXO transcription factor DAF-16 to enhance survival of insulin-like receptor mutant, daf-2 In all, this study shows that PD collagens regulate cuticle permeability by maintaining the structure of C elegans cuticle and thus provide protection against exogenous toxins

Genetic variation in aneuploidy prevalence and tolerance across Saccharomyces cerevisiae lineages.

Abstract: Individuals carrying an aberrant number of chromosomes can vary widely in their expression of aneuploidy phenotypes. A major unanswered question is the degree to which an individual's genetic makeup influences its tolerance of karyotypic imbalance. Here we investigated within-species variation in aneuploidy prevalence and tolerance, using Saccharomyces cerevisiae as a model for eukaryotic biology. We analyzed genotypic and phenotypic variation recently published for over 1,000 S. cerevisiae strains spanning dozens of genetically defined clades and ecological associations. Our results show that the prevalence of chromosome gain and loss varies by clade and can be better explained by differences in genetic background than ecology. The relationships between lineages with high aneuploidy frequencies suggest that increased aneuploidy prevalence emerged multiple times in S. cerevisiae evolution. Separate from aneuploidy prevalence, analyzing growth phenotypes revealed that some genetic backgrounds-such as the European Wine lineage-show fitness costs in aneuploids compared to euploids, whereas other clades with high aneuploidy frequencies show little evidence of major deleterious effects. Our analysis confirms that chromosome gain can produce phenotypic benefits, which could influence evolutionary trajectories. These results have important implications for understanding genetic variation in aneuploidy prevalence in health, disease, and evolution.

Evaluating human autosomal loci for sexually antagonistic viability selection in two large biobanks

Abstract: Sex and sexual differentiation are pervasive across the tree of life Because females and males often have substantially different functional requirements, we expect selection to differ between the sexes Recent studies in diverse species, including humans, suggest that sexually antagonistic viability selection creates allele frequency differences between the sexes at many different loci However, theory and population-level simulations indicate that sex-specific differences in viability would need to be very large to produce and maintain reported levels of between-sex allelic differentiation We address this contradiction between theoretical predictions and empirical observations by evaluating evidence for sexually antagonistic viability selection on autosomal loci in humans using the largest cohort to date (UK Biobank, n = 487,999) along with a second large, independent cohort (BioVU, n = 93,864) We performed association tests between genetically ascertained sex and autosomal loci Although we found dozens of genome-wide significant associations, none replicated across cohorts Moreover, closer inspection revealed that all associations are likely due to cross-hybridization with sex chromosome regions during genotyping We report loci with potential for mis-hybridization found on commonly used genotyping platforms that should be carefully considered in future genetic studies of sex-specific differences Despite being well powered to detect allele frequency differences of up to 08% between the sexes, we do not detect clear evidence for this signature of sexually antagonistic viability selection on autosomal variation These findings suggest a lack of strong ongoing sexually antagonistic viability selection acting on single locus autosomal variation in humans

Prey sensing and response in a nematode-trapping fungus is governed by the MAPK pheromone response pathway

Abstract: Detection of surrounding organisms in the environment plays a major role in the evolution of interspecies interactions, such as predator-prey relationships. Nematode-trapping fungi (NTF) are predators that develop specialized trap structures to capture, kill, and consume nematodes when food sources are limited. Despite the identification of various factors that induce trap morphogenesis, the mechanisms underlying the differentiation process have remained largely unclear. Here, we demonstrate that the highly conserved pheromone-response MAPK pathway is essential for sensing ascarosides, a conserved molecular signature of nemaotdes, and is required for the predatory lifestyle switch in the NTF Arthrobotrys oligospora. Gene deletion of STE7 (MAPKK) and FUS3 (MAPK) abolished nematode-induced trap morphogenesis and conidiation and impaired the growth of hyphae. The conserved transcription factor Ste12 acting downstream of the pheromone-response pathway also plays a vital role in the predation of A. oligospora. Transcriptional profiling of a ste12 mutant identified a small subset of genes with diverse functions that are Ste12 dependent and could trigger trap differentiation. Our work has revealed that A. oligospora perceives and interprets the ascarosides produced by nematodes via the conserved pheromone signaling pathway in fungi, providing molecular insights into the mechanisms of communication between a fungal predator and its nematode prey.

The neural crest cell hypothesis: no unified explanation for domestication.

Abstract: A few years ago, Wilkins et al. (2014) advanced the neural crest cell hypothesis of domestication as an explanation for the “domestication syndrome” in animals, which refers to a collection of characteristic traits associated with domestication. Since then, the neural crest cell hypothesis has risen to become arguably the most popular explanation for the genetics of the initial phase of the domestication process (Zeder 2017; Thomas and Kirby 2018; Kikusui et al. 2019; Zanella et al. 2019). Neural crest cells contribute to many different parts of the vertebrate body. They are the origin of most of the peripheral nervous system including parasympathetic and sympathetic ganglia, the enteric nervous system and sensory ganglia; sensory receptor cells in the skin; pigment cells, except in the retina; endocrine cells in the adrenal medulla and thyroid; the frontal part of the head, including bone, connective tissues, and muscle; and parts of the cardiovascular system (reviewed by Dupin et al. 2006). The neural crest cell hypothesis was motivated by a search for common factors between tameness and other domestication syndrome traits, and an apparent similarity between domestic animals and humans with neurocristopathies, posited by Wilkins et al. (2014). They hypothesized that if tameness is mainly caused by changes in the adrenal glands, selection on tameness might change neural crest development to cause correlated changes such as brain size reduction, floppy ears, altered facial shape, and altered pigmentation. The hypothesis entails that a reduction in neural crest cell proliferation and migration is a core genetic mechanism of early domestication. This implies that the genetic architecture of domestication is by necessity pleiotropic, with variants in genes affecting neural crest development and distribution causing multiple domestication traits. The authors also suggest that neural crest cell function is polygenic, affected by many genetic variants with individually small effects. Furthermore, they have suggested that it involves epigenetic inheritance, genetic assimilation, or epistatic interaction between genes where changes to the neural crest may potentiate later evolution of other domestication traits (Wilkins 2020). Because pleiotropy is at the core of the neural crest cell hypothesis of domestication, we need to clarify what we mean by this term. Paaby and Rockman (2013) distinguish between when genes have multiple functions, for example in different pathways, reactions or protein–protein interactions, and when alleles have effects on multiple traits, for example, by causing a heritable syndrome. As this distinction pertains to the neural crest cell hypothesis, many genes involved in the neural crest are pleiotropic in the sense that they participate in multiple developmental processes. However, for the neural crest cell hypothesis to work, there also have to be pleiotropic alleles, selected during domestication, that affect multiple domestication traits through their effect on the neural crest. The development of neural crest cells is regulated by a complex gene regulatory network that spans multiple cell types and temporal phases (reviewed by Betancur et al. 2010; Rogers et al. 2012). Neural crest cells originate at the border of the neural plate. After induction, they undergo an epithelial-tomesenchymal transition (where they lose their epithelial properties), delaminate (split off) from the neural tube, and migrate to their target locations where they differentiate. Neural crest induction happens in response to secreted signaling molecules [including Bone morphogenetic proteins (BMPs), Fibroblast growth factors (FGFs), and Wnt ligands] secreted by surrounding tissues, and Notch signaling, which is based on cell–cell contact. These signals lead to the expression of neural plate border specifiers (homeobox genes including Msx1 and Pax3 and zinc finger Zic genes), a group of transcription factors that trigger neural crest specifiers. Neural crest specifiers are the key transcription factors expressed in neural crest cells themselves before and during migration (including Snai1/2, Id, cMyc, Sox9, and Sox10). Furthermore, there is a set of genes (including FoxD3, Snai1/2, and Sox5) that regulate how neural crest cells delaminate and go through epithelial-to-mesenchymal transition, needed for them to migrate. This process involves changes to the extracellular matrix, and regulation of cell adhesion molecules including cadherins and integrins. When arriving at their destination, neural crest cells typically stop expressing neural crest specifiers, but some of them keep being expressed and serve to regulate differentiation (e.g., Sox10 in the peripheral nervous system and in melanocytes). Pleiotropic genetic variants acting on neural crest cell development, which is what the neural crest cell hypothesis predicts, could affect any of these processes, up until but likely not

Pleiotropy or linkage? Their relative contributions to the genetic correlation of quantitative traits and detection by multitrait GWA studies.

Abstract: Genetic correlations between traits may cause correlated responses to selection. Previous models described the conditions under which genetic correlations are expected to be maintained. Selection, mutation, and migration are all proposed to affect genetic correlations, regardless of whether the underlying genetic architecture consists of pleiotropic or tightly linked loci affecting the traits. Here, we investigate the conditions under which pleiotropy and linkage have different effects on the genetic correlations between traits by explicitly modeling multiple genetic architectures to look at the effects of selection strength, degree of correlational selection, mutation rate, mutational variance, recombination rate, and migration rate. We show that at mutation-selection(-migration) balance, mutation rates differentially affect the equilibrium levels of genetic correlation when architectures are composed of pairs of physically linked loci compared to architectures of pleiotropic loci. Even when there is perfect linkage (no recombination within pairs of linked loci), a lower genetic correlation is maintained than with pleiotropy, with a lower mutation rate leading to a larger decrease. These results imply that the detection of causal loci in multitrait association studies will be affected by the type of underlying architectures, whereby pleiotropic variants are more likely to be underlying multiple detected associations. We also confirm that tighter linkage between nonpleiotropic causal loci maintains higher genetic correlations at the traits and leads to a greater proportion of false positives in association analyses.

An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans.

Abstract: The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies

Abstract: The eyespot patterns found on the wings of nymphalid butterflies are novel traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential not only for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serially homologous segments along the anterior-posterior axis of insects, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development followed by a later, partially redundant, co-option of Antp into the same network.

Optimal breeding-value prediction using a sparse selection index.

Abstract: Genomic prediction uses DNA sequences and phenotypes to predict genetic values. In homogeneous populations, theory indicates that the accuracy of genomic prediction increases with sample size. However, differences in allele frequencies and linkage disequilibrium patterns can lead to heterogeneity in SNP effects. In this context, calibrating genomic predictions using a large, potentially heterogeneous, training data set may not lead to optimal prediction accuracy. Some studies tried to address this sample size/homogeneity trade-off using training set optimization algorithms; however, this approach assumes that a single training data set is optimum for all individuals in the prediction set. Here, we propose an approach that identifies, for each individual in the prediction set, a subset from the training data (i.e., a set of support points) from which predictions are derived. The methodology that we propose is a sparse selection index (SSI) that integrates selection index methodology with sparsity-inducing techniques commonly used for high-dimensional regression. The sparsity of the resulting index is controlled by a regularization parameter (λ); the G-Best Linear Unbiased Predictor (G-BLUP) (the prediction method most commonly used in plant and animal breeding) appears as a special case which happens when λ = 0. In this study, we present the methodology and demonstrate (using two wheat data sets with phenotypes collected in 10 different environments) that the SSI can achieve significant (anywhere between 5 and 10%) gains in prediction accuracy relative to the G-BLUP.

Haplotype reconstruction in connected tetraploid F1 populations.

Abstract: In diploid species, many multiparental populations have been developed to increase genetic diversity and quantitative trait loci (QTL) mapping resolution. In these populations, haplotype reconstruction has been used as a standard practice to increase the power of QTL detection in comparison with the marker-based association analysis. However, such software tools for polyploid species are few and limited to a single biparental F1 population. In this study, a statistical framework for haplotype reconstruction has been developed and implemented in the software PolyOrigin for connected tetraploid F1 populations with shared parents, regardless of the number of parents or mating design. Given a genetic or physical map of markers, PolyOrigin first phases parental genotypes, then refines the input marker map, and finally reconstructs offspring haplotypes. PolyOrigin can utilize single nucleotide polymorphism (SNP) data coming from arrays or from sequence-based genotyping; in the latter case, bi-allelic read counts can be used (and are preferred) as input data to minimize the influence of genotype calling errors at low depth. With extensive simulation we show that PolyOrigin is robust to the errors in the input genotypic data and marker map. It works well for various population designs with ≥30 offspring per parent and for sequences with read depth as low as 10x. PolyOrigin was further evaluated using an autotetraploid potato dataset with a 3 × 3 half-diallel mating design. In conclusion, PolyOrigin opens up exciting new possibilities for haplotype analysis in tetraploid breeding populations.

De novo assembly and delivery to mouse cells of a 101 kb functional human gene.

Abstract: Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

Meta-analysis identifies pleiotropic loci controlling phenotypic trade-offs in sorghum.

Abstract: Community association populations are composed of phenotypically and genetically diverse accessions. Once these populations are genotyped, the resulting marker data can be reused by different groups investigating the genetic basis of different traits. Because the same genotypes are observed and scored for a wide range of traits in different environments, these populations represent a unique resource to investigate pleiotropy. Here, we assembled a set of 234 separate trait datasets for the Sorghum Association Panel, a group of 406 sorghum genotypes widely employed by the sorghum genetics community. Comparison of genome-wide association studies (GWAS) conducted with two independently generated marker sets for this population demonstrate that existing genetic marker sets do not saturate the genome and likely capture only 35-43% of potentially detectable loci controlling variation for traits scored in this population. While limited evidence for pleiotropy was apparent in cross-GWAS comparisons, a multivariate adaptive shrinkage approach recovered both known pleiotropic effects of existing loci and new pleiotropic effects, particularly significant impacts of known dwarfing genes on root architecture. In addition, we identified new loci with pleiotropic effects consistent with known trade-offs in sorghum development. These results demonstrate the potential for mining existing trait datasets from widely used community association populations to enable new discoveries from existing trait datasets as new, denser genetic marker datasets are generated for existing community association populations.

The rates of introgression and barriers to genetic exchange between hybridizing species: sex chromosomes vs autosomes.

Abstract: Interspecific crossing experiments have shown that sex chromosomes play a major role in reproductive isolation between many pairs of species. However, their ability to act as reproductive barriers, which hamper interspecific genetic exchange, has rarely been evaluated quantitatively compared to Autosomes. This genome-wide limitation of gene flow is essential for understanding the complete separation of species, and thus speciation. Here, we develop a mainland-island model of secondary contact between hybridizing species of an XY (or ZW) sexual system. We obtain theoretical predictions for the frequency of introgressed alleles, and the strength of the barrier to neutral gene flow for the two types of chromosomes carrying multiple interspecific barrier loci. Theoretical predictions are obtained for scenarios where introgressed alleles are rare. We show that the same analytical expressions apply for sex chromosomes and autosomes, but with different sex-averaged effective parameters. The specific features of sex chromosomes (hemizygosity and absence of recombination in the heterogametic sex) lead to reduced levels of introgression on the X (or Z) compared to autosomes. This effect can be enhanced by certain types of sex-biased forces, but it remains overall small (except when alleles causing incompatibilities are recessive). We discuss these predictions in the light of empirical data comprising model-based tests of introgression and cline surveys in various biological systems.

Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans.

Abstract: Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans' ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.

Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging.

Abstract: Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized (Cno) Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (Cno CAS9 or "CnoCAS9") reliably enabled genome editing in the widely used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

In vivo tissue-specific chromatin profiling in Drosophila melanogaster using GFP-tagged nuclei.

Abstract: The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.

Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.

Abstract: We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663.

Sweeps in time: leveraging the joint distribution of branch lengths

Abstract: Current methods of identifying positively selected regions in the genome are limited in two key ways: the underlying models cannot account for the timing of adaptive events and the comparison between models of selective sweeps and sequence data is generally made via simple summaries of genetic diversity. Here, we develop a tractable method of describing the effect of positive selection on the genealogical histories in the surrounding genome, explicitly modeling both the timing and context of an adaptive event. In addition, our framework allows us to go beyond analyzing polymorphism data via the site frequency spectrum or summaries thereof and instead leverage information contained in patterns of linked variants. Tests on both simulations and a human data example, as well as a comparison to SweepFinder2, show that even with very small sample sizes, our analytic framework has higher power to identify old selective sweeps and to correctly infer both the time and strength of selection. Finally, we derived the marginal distribution of genealogical branch lengths at a locus affected by selection acting at a linked site. This provides a much-needed link between our analytic understanding of the effects of sweeps on sequence variation and recent advances in simulation and heuristic inference procedures that allow researchers to examine the sequence of genealogical histories along the genome.