Genome-scale analysis of interactions between genetic perturbations and natural variation.

Interactions between genetic perturbations and segregating loci can cause perturbations to show different phenotypic effects across genetically distinct individuals. To study these interactions on a genome scale in many individuals, we used combinatorial DNA barcode sequencing to measure the fitness effects of 7,700 CRISPRi perturbations targeting 1,712 distinct genes in 169 yeast cross progeny (or segregants). We identified 460 genes whose perturbation has different effects across segregants. Several factors caused perturbations to show variable effects, including baseline segregant fitness, the mean effect of a perturbation across segregants, and interacting loci. We mapped 234 interacting loci and found four hub loci that interact with many different perturbations. Perturbations that interact with a given hub exhibit similar epistatic relationships with the hub and show enrichment for cellular processes that may mediate these interactions. These results suggest that an individual's response to perturbations is shaped by a network of perturbation-locus interactions that cannot be measured by approaches that examine perturbations or natural variation alone.

Building synthetic chromosomes from natural DNA.

De novo chromosome synthesis is costly and time-consuming, limiting its use in research and biotechnology. Building synthetic chromosomes from natural components is an unexplored alternative with many potential applications. In this paper, we report CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), a method for constructing synthetic chromosomes from natural components in yeast. CReATiNG entails cloning segments of natural chromosomes and then programmably assembling them into synthetic chromosomes that can replace the native chromosomes in cells. We use CReATiNG to synthetically recombine chromosomes between strains and species, to modify chromosome structure, and to delete many linked, non-adjacent regions totaling 39% of a chromosome. The multiplex deletion experiment reveals that CReATiNG also enables recovery from flaws in synthetic chromosome design via recombination between a synthetic chromosome and its native counterpart. CReATiNG facilitates the application of chromosome synthesis to diverse biological problems.

Building synthetic chromosomes from natural DNA.

De novo chromosome synthesis is costly and time-consuming, limiting its use in research and biotechnology. Building synthetic chromosomes from natural components is an unexplored alternative with many potential applications. In this paper, we report CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), a method for constructing synthetic chromosomes from natural components in yeast. CReATiNG entails cloning segments of natural chromosomes and then programmably assembling them into synthetic chromosomes that can replace the native chromosomes in cells. We used CReATiNG to synthetically recombine chromosomes between strains and species, to modify chromosome structure, and to delete many linked, non-adjacent regions totaling 39% of a chromosome. The multiplex deletion experiment revealed that CReATiNG also enables recovery from flaws in synthetic chromosome design via recombination between a synthetic chromosome and its native counterpart. CReATiNG facilitates the application of chromosome synthesis to diverse biological problems.

Complex genetics cause and constrain fungal persistence in different parts of the mammalian body.

Determining how genetic polymorphisms enable certain fungi to persist in mammalian hosts can improve understanding of opportunistic fungal pathogenesis, a source of substantial human morbidity and mortality. We examined the genetic basis of fungal persistence in mice using a cross between a clinical isolate and the lab reference strain of the budding yeast Saccharomyces cerevisiae. Employing chromosomally encoded DNA barcodes, we tracked the relative abundances of 822 genotyped, haploid segregants in multiple organs over time and performed linkage mapping of their persistence in hosts. Detected loci showed a mix of general and antagonistically pleiotropic effects across organs. General loci showed similar effects across all organs, while antagonistically pleiotropic loci showed contrasting effects in the brain vs the kidneys, liver, and spleen. Persistence in an organ required both generally beneficial alleles and organ-appropriate pleiotropic alleles. This genetic architecture resulted in many segregants persisting in the brain or in nonbrain organs, but few segregants persisting in all organs. These results show complex combinations of genetic polymorphisms collectively cause and constrain fungal persistence in different parts of the mammalian body.

The interplay of additivity, dominance, and epistasis on fitness in a diploid yeast cross.

In diploid species, genetic loci can show additive, dominance, and epistatic effects. To characterize the contributions of these different types of genetic effects to heritable traits, we use a double barcoding system to generate and phenotype a panel of ~200,000 diploid yeast strains that can be partitioned into hundreds of interrelated families. This experiment enables the detection of thousands of epistatic loci, many whose effects vary across families. Here, we show traits are largely specified by a small number of hub loci with major additive and dominance effects, and pervasive epistasis. Genetic background commonly influences both the additive and dominance effects of loci, with multiple modifiers typically involved. The most prominent dominance modifier in our data is the mating locus, which has no effect on its own. Our findings show that the interplay between additivity, dominance, and epistasis underlies a complex genotype-to-phenotype map in diploids.

Genetic basis of a spontaneous mutation's expressivity.

Genetic background often influences the phenotypic consequences of mutations, resulting in variable expressivity. How standing genetic variants collectively cause this phenomenon is not fully understood. Here, we comprehensively identify loci in a budding yeast cross that impact the growth of individuals carrying a spontaneous missense mutation in the nuclear-encoded mitochondrial ribosomal gene MRP20. Initial results suggested that a single large effect locus influences the mutation's expressivity, with 1 allele causing inviability in mutants. However, further experiments revealed this simplicity was an illusion. In fact, many additional loci shape the mutation's expressivity, collectively leading to a wide spectrum of mutational responses. These results exemplify how complex combinations of alleles can produce a diversity of qualitative and quantitative responses to the same mutation.

Transcriptomic bases of a polyphenism.

Polyphenism-in which multiple distinct phenotypes are produced from a single genotype owing to differing environmental conditions-is commonplace, but its molecular bases are poorly understood. Here, we examine the transcriptomic bases of a polyphenism in Mexican spadefoot toads (Spea multiplicata). Depending on their environment, their tadpoles develop into either a default "omnivore" morph or a novel "carnivore" morph. We compared patterns of gene expression among sibships that exhibited high versus low production of carnivores when reared in conditions that induce the carnivore morph versus those that do not. We found that production of the novel carnivore morph actually involved changes in fewer genes than did the maintenance of the default omnivore morph in the inducing environment. However, only body samples showed this pattern; head samples showed the opposite pattern. We also found that changes to lipid metabolism (especially cholesterol biosynthesis) and peroxisome contents and function might be crucial for establishing and maintaining differences between the morphs. Thus, our findings suggest that carnivore phenotype might have originally evolved following the breakdown of robustness mechanisms that maintain the default omnivore phenotype, and that the carnivore morph is developmentally regulated by lipid metabolism and peroxisomal form, function, and/or signaling. This study also serves as a springboard for further exploration into the nature and causes of plasticity in an emerging model system.

Evolution in Long-Term Stationary-Phase Batch Culture: Emergence of Divergent Escherichia coli Lineages over 1,200 Days.

In natural environments, bacteria survive conditions of starvation and stress. Long-term batch cultures are an excellent laboratory system to study adaptation during nutrient stress because cells can incubate for months to years without the addition of nutrients. During long-term batch culture, cells adapt to acquire energy from cellular detritus, creating a complex and dynamic environment for mutants of increased relative fitness to exploit. Here, we analyzed the genomes of 1,117 clones isolated from a single long-term batch culture incubated for 1,200 days. A total of 679 mutations included single nucleotide polymorphisms, indels, mobile genetic element movement, large deletions up to 64 kbp, and amplifications up to ∼500 kbp. During the 3.3-year incubation, two main lineages diverged, evolving continuously. At least twice, a previously fixed mutation reverted back to the wild-type allele, suggesting beneficial mutations may later become maladaptive due to the dynamic environment and changing selective pressures. Most of the mutated genes encode proteins involved in metabolism, transport, or transcriptional regulation. Clones from the two lineages are physiologically distinct, based on outgrowth in fresh medium and competition against the parental strain. Similar population dynamics and mutations in hfq, rpoS, paaX, lrp, sdhB, and dtpA were detected in three additional parallel populations sequenced through day 60, providing evidence for positive selection. These data provide new insight into the population structure and mutations that may be beneficial during periods of starvation in evolving bacterial communities.IMPORTANCE Bacteria have remarkable metabolic capabilities and adaptive plasticity, enabling them to survive in changing environments. In nature, bacteria spend a majority of their time in a state of slow growth or maintenance, scavenging nutrients for survival. Here, a population of Escherichia coli cells was incubated for 1,200 days in long-term batch culture, without the addition of new medium, requiring cells to continuously recycle nutrients. Whole-genome resequencing of cells from the evolving population identified two dominant subpopulations that coexisted while continuously acquiring and fixing new mutations. The population dynamics and alleles identified provide insight into adaptation to nutrient stress. Elucidating mechanisms that allow bacteria to adapt through cycles of feast and famine deepens our understanding of their survival mechanisms in nature.

The complex role of genetic background in shaping the effects of spontaneous and induced mutations.

Spontaneous and induced mutations frequently show different phenotypic effects across genetically distinct individuals. It is generally appreciated that these background effects mainly result from genetic interactions between the mutations and segregating loci. However, the architectures and molecular bases of these genetic interactions are not well understood. Recent work in a number of model organisms has tried to advance knowledge of background effects both by using large-scale screens to find mutations that exhibit this phenomenon and by identifying the specific loci that are involved. Here, we review this body of research, emphasizing in particular the insights it provides into both the prevalence of background effects across different mutations and the mechanisms that cause these background effects.

Building genomes to understand biology.

Genetic manipulation is one of the central strategies that biologists use to investigate the molecular underpinnings of life and its diversity. Thus, advances in genetic manipulation usually lead to a deeper understanding of biological systems. During the last decade, the construction of chromosomes, known as synthetic genomics, has emerged as a novel approach to genetic manipulation. By facilitating complex modifications to chromosome content and structure, synthetic genomics opens new opportunities for studying biology through genetic manipulation. Here, we discuss different classes of genetic manipulation that are enabled by synthetic genomics, as well as biological problems they each can help solve.

Genome of Spea multiplicata, a Rapidly Developing, Phenotypically Plastic, and Desert-Adapted Spadefoot Toad.

Frogs and toads (anurans) are widely used to study many biological processes. Yet, few anuran genomes have been sequenced, limiting research on these organisms. Here, we produce a draft genome for the Mexican spadefoot toad, Spea multiplicata, which is a member of an unsequenced anuran clade. Atypically for amphibians, spadefoots inhabit deserts. Consequently, they possess many unique adaptations, including rapid growth and development, prolonged dormancy, phenotypic (developmental) plasticity, and adaptive, interspecies hybridization. We assembled and annotated a 1.07 Gb Sp. multiplicata genome containing 19,639 genes. By comparing this sequence to other available anuran genomes, we found gene amplifications in the gene families of nodal, hyas3, and zp3 in spadefoots, and obtained evidence that anuran genome size differences are partially driven by variability in intergenic DNA content. We also used the genome to identify genes experiencing positive selection and to study gene expression levels in spadefoot hybrids relative to their pure-species parents. Completion of the Sp. multiplicata genome advances efforts to determine the genetic bases of spadefoots' unique adaptations and enhances comparative genomic research in anurans.

Variation in hybrid gene expression: Implications for the evolution of genetic incompatibilities in interbreeding species.

Interbreeding species often produce low-fitness hybrids due to genetic incompatibilities between parental genomes. Whether these incompatibilities reflect fixed allelic differences between hybridizing species, or, alternatively, standing variants that segregate within them, remains unknown for many natural systems. Yet, evaluating these alternatives is important for understanding the origins and nature of species boundaries. We examined these alternatives using spadefoot toads (genus Spea), which naturally hybridize. Specifically, we contrasted patterns of gene expression in hybrids relative to pure-species types in experimentally produced tadpoles from allopatric parents versus those from sympatric parents. We evaluated the prediction that segregating variation should result in gene expression differences between hybrids derived from sympatric parents versus hybrids derived from allopatric parents, and found that 24% of the transcriptome showed such differences. Our results further suggest that gene expression in hybrids has evolved in sympatry owing to evolutionary pressures associated with ongoing hybridization. Although we did not measure hybrid incompatibilities directly, we discuss the implications of our findings for understanding the nature of hybrid incompatibilities, how they might vary across populations over time, and the resulting effects on the evolutionary maintenance - or breakdown - of reproductive barriers between species.

Layers of Cryptic Genetic Variation Underlie a Yeast Complex Trait.

Cryptic genetic variation may be an important contributor to heritable traits, but its extent and regulation are not fully understood. Here, we investigate the cryptic genetic variation underlying a Saccharomyces cerevisiae colony phenotype that is typically suppressed in a cross of the laboratory strain BY4716 (BY) and a derivative of the clinical isolate 322134S (3S). To do this, we comprehensively dissect the trait's genetic basis in the BYx3S cross in the presence of three different genetic perturbations that enable its expression. This allows us to detect and compare the specific loci that interact with each perturbation to produce the trait. In total, we identify 21 loci, all but one of which interact with just a subset of the perturbations. Beyond impacting which loci contribute to the trait, the genetic perturbations also alter the extent of additivity, epistasis, and genotype-environment interaction among the detected loci. Additionally, we show that the single locus interacting with all three perturbations corresponds to the coding region of the cell surface gene FLO11 While nearly all of the other remaining loci influence FLO11 transcription in cis or trans, the perturbations tend to interact with loci in different pathways and subpathways. Our work shows how layers of cryptic genetic variation can influence complex traits. Here, these layers mainly represent different regulatory inputs into the transcription of a single key gene.

The complex underpinnings of genetic background effects.

Genetic interactions between mutations and standing polymorphisms can cause mutations to show distinct phenotypic effects in different individuals. To characterize the genetic architecture of these so-called background effects, we genotype 1411 wild-type and mutant yeast cross progeny and measure their growth in 10 environments. Using these data, we map 1086 interactions between segregating loci and 7 different gene knockouts. Each knockout exhibits between 73 and 543 interactions, with 89% of all interactions involving higher-order epistasis between a knockout and multiple loci. Identified loci interact with as few as one knockout and as many as all seven knockouts. In mutants, loci interacting with fewer and more knockouts tend to show enhanced and reduced phenotypic effects, respectively. Cross-environment analysis reveals that most interactions between the knockouts and segregating loci also involve the environment. These results illustrate the complicated interactions between mutations, standing polymorphisms, and the environment that cause background effects.

The Stress-Inducible Peroxidase TSA2 Underlies a Conditionally Beneficial Chromosomal Duplication in Saccharomyces cerevisiae.

Although chromosomal duplications are often deleterious, in some cases they enhance cells' abilities to tolerate specific genetic or environmental challenges. Identifying the genes that confer these conditionally beneficial effects to particular chromosomal duplications can improve our understanding of the genetic and molecular mechanisms that enable certain aneuploidies to persist in cell populations and contribute to disease and evolution. Here, we perform a screen for spontaneous mutations that improve the tolerance of haploid Saccharomyces cerevisiae to hydrogen peroxide. Chromosome IV duplication is the most frequent mutation, as well as the only change in chromosomal copy number seen in the screen. Using a genetic mapping strategy that involves systematically deleting segments of a duplicated chromosome, we show that the chromosome IV's duplication effect is largely due to the generation of a second copy of the stress-inducible cytoplasmic thioredoxin peroxidase TSA2 Our findings add to a growing body of literature that shows the conditionally beneficial effects of chromosomal duplication are typically mediated by a small number of genes that enhance tolerance to specific stresses when their copy numbers are increased.

Genetic Dissection of Heritable Traits in Yeast Using Bulk Segregant Analysis.

Bulk segregant analysis (BSA) is commonly used to determine the genetic basis of complex traits in yeast. This technique involves phenotyping progeny from a cross and then selectively genotyping pooled subsets of offspring with extreme phenotypes. Analysis of these genotype data can identify loci that show skewed allele frequencies in a group of phenotypically extreme individuals and that are likely to contribute to a trait. BSA can be applied to diverse strain crosses, including ones involving nonreference isolates. Further, given the high throughput of next-generation sequencing, it is possible to conduct many BSA experiments in parallel. Here, we present a BSA protocol for the generation of recombinant cross progeny. We then describe general BSA strategies for conducting phenotyping, causal loci detection, and candidate gene identification in a statistically powerful manner.

Genetic Analysis of Complex Traits in Saccharomyces cerevisiae.

Defining the relationship between genotype and phenotype is a central challenge in biology. A powerful approach to this problem is to determine the genetic architecture and molecular basis of phenotypic differences among genetically diverse individuals. Saccharomyces cerevisiae is an important model system for such work. Current genetic mapping approaches for this species exploit high-throughput phenotyping and sequencing to facilitate the detection of a large fraction of the genomic loci that underlie trait variation among isolates. Once identified, several methods exist to determine the specific genes and genetic variants that underlie these loci and cause phenotypic variations. In this introduction, we provide a brief overview of research on complex traits in yeast and discuss different genetic mapping approaches applied to yeast studies.