G3 Genes|Genomes|Genetics

Growth rate and body size are complex traits that contribute to the fitness of organisms. The identification of loci that underlie differences in these traits provides insights into the genetic contributions to development. Leveraging Caenorhabditis elegans as a tractable metazoan model for quantitative genetics, we can identify genomic regions that underlie differences in growth. We measured post-embryonic growth of the laboratory-adapted wild-type strain (N2) and a wild strain from Hawaii (CB4856), and found differences in body size. Using linkage mapping, we identified three distinct quantitative trait loci (QTL) on chromosomes IV, V, and X that are associated with variation in body size. We further examined these size-associated QTL using chromosome substitution strains and near-isogenic lines, and validated the chromosome X QTL. Additionally, we generated a list of candidate genes for the chromosome X QTL. These genes could potentially contribute to differences in animal growth and should be evaluated in subsequent studies. Our work reveals the genetic architecture underlying animal growth variation and highlights the genetic complexity of body size in C. elegans natural populations.

1.The Cell Division Cycle and Apoptosis Regulator (CCAR) protein family members are putative transcription regulators that have been characterized for modulating the cell cycle, apoptosis, metabolism, and the heat shock response. Mammals have two CCAR family members, CCAR1 and CCAR2/DBC1, that evolved from the founding family member CCAR-1 that is expressed in Caenorhabditis elegans. Mammalian CCAR2, the most well-studied family member, has been shown to regulate genes involved in metabolism in cultured cells. However, the regulation of gene expression by CCAR family members at an organismal level is unknown. Here, we use whole transcriptome RNA sequencing to examine the effects of CCAR-1 on gene expression in Caenorhabditis elegans. We show that CCAR-1 regulates germline transcription, reproduction, lifespan, and DNA-damage induced apoptosis. This study shows the role of CCAR-1 in vital physiological functions in the C. elegans germline that have not been investigated before.

Host selection is often required to foster beneficial microbial symbionts and suppress deleterious pathogens. In animals, the host immune system is at the center of this relationship. Failed host immune system-microbial interactions can result in a persistent inflammatory response in which the immune system indiscriminately attacks resident microbes, and at times the host cells themselves, leading to diseases such as Ulcerative Colitis, Crohns Disease, and Psoriasis. Host genetic variation has been linked to both microbiome diversity and to severity of such inflammatory disease states in humans. However, the microbiome and inflammatory states manifest as quantitative traits, which encompass many genes interacting with one another and the environment. The mechanistic relationships among all of these interacting components are still not clear. Developing natural genetic models of host-microbe interactions is therefore fundamental to understanding the complex genetics of these and other diseases. Threespine stickleback (Gasterosteus aculeatus) fish are a tractable model for attacking this problem because of abundant population-level genetic and phenotypic variation in the gut inflammatory response. Previous work in our laboratory identified genetically divergent stickleback populations exhibiting differences in intestinal neutrophil activity. We took advantage of this diversity to genetically map variation in an emblematic element of gut inflammation - intestinal neutrophil recruitment - using an F2-intercross mapping framework. We identified three regions of the genome associated with increased intestinal inflammation containing several promising candidate genes. Within these regions we found candidates in the Coagulation/Complement System, NFkB and MAPK pathways along with several genes associated with neurodegenerative diseases commonly accompanying intestinal inflammation as a secondary symptom. These findings highlight the utility of using naturally genetically diverse evolutionary mutant models such as threespine stickleback to better understand interactions among host genetic diversity and microbiome variation in health and disease states.

Power analyses are often used to determine the number of animals required for a genome wide association analysis (GWAS). These analyses are typically intended to estimate the sample size needed for at least one locus to exceed a genome-wide significance threshold. A related question that is less commonly considered is the number of significant loci that will be discovered with a given sample size. We used simulations based on a real dataset that consisted of 3,173 male and female adult N/NIH heterogeneous stock (HS) rats to explore the relationship between sample size and the number of significant loci discovered. Our simulations examined the number of loci identified in sub-samples of the full dataset. The sub-sampling analysis was conducted for four traits with low (0.15 {+/-} 0.03), medium (0.31 {+/-} 0.03 and 0.36 {+/-} 0.03) and high (0.46 {+/-} 0.03) SNP-based heritabilities. For each trait, we sub-sampled the data 100 times at different sample sizes (500, 1,000, 1,500, 2,000, and 2,500). We observed an exponential increase in the number of significant loci with larger sample sizes. Our results are consistent with similar observations in human GWAS and imply that future rodent GWAS should use sample sizes that are significantly larger than those needed to obtain a single significant result.

Quantitative genetics seeks to understand the role of allelic variation in trait differences. Introgression lines (ILs) contain a single genetic locus introgressed into another genetic background, and are one of the most powerful quantitative trait locus (QTL) mapping designs. However, albeit useful for QTL discovery, this homogenous background confounds genetic interactions. Here, we created an IL population with N2 segments in a CB4856 background (ILCB4856), reciprocal to an N2 background with CB4856 introgressions population (ILN2). The ILCB4856 panel comprises a population of 145 strains with sequencing confirmed N2 introgressions in a CB4856 background. A core set of 87 strains covering the entire genome was selected. We present three experiments demonstrating the power of the reciprocal IL panels. First, we performed QTL mapping identifying new regions associated with lifespan. Second, the existence of opposite-effect loci regulating heat-stress survival is demonstrated. Third, by combining ILN2 and ILCB4856 strains, an interacting expression QTL was uncovered. In conclusion, the reciprocal IL panels are a unique and ready-to-use resource to identify, resolve, and refine complex trait architectures in C. elegans.

Important uncertainties persist regarding the genetic architecture of adaptive trait evolution in natural populations, including the number of genetic variants involved, whether they are drawn from standing genetic variation, and whether directional selection drives them to complete fixation. Here, we take advantage of a unique natural population of Drosophila melanogaster from the Ethiopian highlands, which has evolved larger body size than any other known population of this species. We apply a bulk segregant quantitative trait locus (QTL) mapping approach to four unique crosses between highland Ethiopian and lowland Zambian populations for both thorax length and wing length. Results indicated a persistently variable genetic basis for these evolved traits (with largely distinct sets of QTLs for each cross), and at least a moderately polygenic architecture with relatively strong effects present. We complemented these mapping experiments with population genetic analyses of QTL regions and gene ontology enrichment analysis, generating strong hypotheses for specific genes and functional processes that may have contributed to these adaptive trait changes. Finally, we find that the genetic architectures our QTL mapping results for size traits mirror those from similar experiments on other recently-evolved traits in this species. Collectively, these studies suggest a recurring pattern of polygenic adaptation in this species, in which causative variants do not approach fixation and moderately strong effect loci are present.

Phenotypic development is regulated by multiple mechanisms that ensure tight control of gene expression. Post-transcriptional regulation, including the silencing or degradation of messenger RNAs by microRNAs (miRNAs), is an important component of this process. Here, we use gain-of-function and loss-of-function screens to examine the effects of miRNAs on cuticular pigmentation in adult Drosophila melanogaster. We found that 48 of 166 miRNAs ectopically expressed in a stripe along the dorsal side of developing flies were each sufficient to affect pigmentation. We also found that 22 of 41 miRNAs competitively inhibited in the same tissue visibly altered pigmentation, showing that they were necessary for adult pigmentation development. For each of the 15 miRNAs with opposing effects in the gain- and loss-of-function screens, computational tools identified possible targets among 93 genes previously reported to affect adult pigmentation. Using cell culture, we found that one of these miRNAs (miR-8) was able to regulate gene expression through 3 UTR sequences from at least three pigmentation genes: ebony, bric-a-brac 1, and bric-a-brac 2. All three of these genes reduce development of black pigments, suggesting that miR-8 coordinately regulates expression of multiple genes with similar effects on pigmentation. These data show that miRNAs are important developmental regulators of body pigmentation, which could also allow them to contribute to pigmentation divergence, as has been shown for miR-193 in butterflies.

Metabolic Syndrome (MetS) risk, driven by genotype-environment interactions like diet, is rising globally. Due to its genetic and environmental complexity, the genetic architecture and interconnected traits underlying MetS is poorly understood. In Drosophila, genotype-by-diet interactions significantly influence MetS-like traits. This study used the Drosophila Synthetic Population Resource to dissect the genetic architecture of both genotypic and genotype-by-diet interaction effects underlying trait variation. The study hypotheses were: 1) Loci responsible for metabolic phenotypic variation should be shared across traits. 2) Genetic loci responsible for plasticity and epistatic interactions for metabolic traits should also be the loci responsible for the main effects. 3) Genes responsible for variation in metabolic traits should share common functions. Using a round-robin crossing scheme and novel analyses, we mapped additive, dominance, and epistatic loci--some diet-specific, others diet-independent. Main-effect and plastic loci were largely distinct, as were epistatic loci from main-effect loci, highlighting that main genetic effects alone will not explain how genetic variants interact with the environment or the genome to influence disease risk. gene-by-diet or gene-by-gene interactions influencing MetS risk. Further, tremendous cryptic genetic variation for metabolic traits is lurking in natural populations. We explored the function of candidate genes from our study empirically and with bioinformatics. While some of the candidate genes might have been expected, most would not have been identified a priori, thus with this study we have identified many new candidate mechanisms contributing to the genetic and genotype-by-diet interaction effects on MetS variance.

The body muscle is an important tissue used in organisms for proper viability and locomotion. Although this tissue is generally well studied and characterized, and many pathways have been elucidated throughout the years, we still lack a comprehensive understanding of its transcriptome and how it controls muscle development and function. Here, we have updated a nuclear FACS sorting-based methodology to isolate and sequence a high-quality muscle transcriptome from C. elegans mixed-stage animals. We have identified 2,848 muscle-specific protein-coding genes, including 78 transcription factors and 206 protein-coding genes containing an RNA binding domain. We studied their interaction network, performed a detailed promoter analysis, and identified novel muscle-specific cis-acting elements. We have also identified 16 high-quality muscle-specific miRNAs, studied their function in vivo using fluorochrome-based analyses, and developed a high-quality C. elegans miRNA Interactome incorporating other muscle-specific datasets produced by our lab and others. Our study expands our understanding of how muscle tissue functions in C. elegans and in turn, provide results that can in the future be applied to humans to study muscular-related diseases.

The maize weevil, Sitophilus zeamais, is a worldwide pest that disproportionately affects subsistence farmers in developing countries. Damage from this pest threatens food security in these communities as widely available and effective control methods are lacking. With advances over the last decade in the development of advanced genetic pest management techniques, addressing pest issues at the ecosystem level as opposed to the farm level may be a possibility. However, pest species selected for genetic management techniques require a well-characterized genome and few genomic tools have been developed for maize weevil. Here, we have measured the genome size and developed the first genetic linkage map for this species. The genome size was determined using flow cytometry as 682 Mb and 674 Mb for females and males, respectively. The linkage map contains 11 linkage groups, which correspond to the 10 autosomes and 1 X-chromosome found in the species and it contains 1,121 SNPs. This linkage map will be useful for assembling a complete genome for maize weevil.

A central challenge of quantitative genetics is partitioning phenotypic variation into genetic and non-genetic components. These non-genetic components are usually interpreted as environmental effects; however, variation between genetically identical individuals in a common environment can still exhibit phenotypic variation. A traits resistance to variation is called robustness, though the genetics underlying it are poorly understood. Accordingly, we performed an association study on a previously studied, whole organism trait: robustness for flight performance. Using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, we surveyed variation across single nucleotide polymorphisms, whole genes, and epistatic interactions to find genetic modifiers robustness for flight performance. There was an abundance of genes involved in the development of sensory organs and processing of external stimuli, supporting previous work that processing proprioceptive cues is important for affecting variation in flight performance. Additionally, we tested insertional mutants for their effect on robustness using candidate genes found to modify flight performance. These results suggest several genes involved in modulating a trait mean are also important for affecting trait variance, or robustness, as well. Article SummaryWe sought to understand the genetic architecture of robustness (variation in a trait caused by non-genetic factors) for flight performance. We used 197 Drosophila Genetic Reference Panel (DGRP) lines to find significant individual variants and pairs of epistatic interactions, many of which were involved in proprioception. Additionally, we validated significant genes identified from a prior study for the mean of flight performance, showing genes affecting trait means may also affect trait robustness.

Drosophila has been a powerful model system for biological studies due to the wide range of genetic tools established for it. Among these tools, Gal4 is the most abundant, offering unparalleled tissue- and developmental stage-specificity for gene manipulation. In comparison, other genetic reagents are far fewer in choices. Here we present a genetic toolkit for converting Gal4 strains into LexA and Flippase transgenes through simple genetic crosses and fluorescence screening. We demonstrate the proof-of-principle by converting ten Gal4 lines that exhibit diverse tissue specificities and examined the activity patterns of the converted LexA and Flippase lines. Gal4-to-LexA and Flp conversion is fast and convenient and should greatly expand the choices of LexA and Flp for binary expression and FRT-based mosaic analysis, respectively, in Drosophila.

The availabilty of reference genomes is accelerating rapidly, making their use in a wide variety of biological research programmes more feasible than ever. However, current Next-Generation Sequencing platforms are limited in the length of reads they are able to produce; requiring the correct order to be determined algorithmically. While there is a potential for errors in assembly algorithims, genetic pedigree data can be used to identify recombination events and, as recombination events are rare locally, test the order of sequences within a genome assembly. We use high-resolution population genomic data to test and compare the assembly quality of the three most recent reference genome assemblies for the western honey bee (Apis mellifera). As a model organism, there are several reference genomes available for A. mellifera with estimated recombination rates ranging from 19 cM/Mb to 37 cM/Mb. We identify a large degree of variation between assemblies and find that at least 20% of the most recent A. mellifera reference genome is mis-assembled. Providing an explanation for the degree of variation in estimated recombination rates and potentially influencing results downstream.

Convenient and economical genotyping methods and simplified bioinformatic workflows are critical for genetic studies and breeding. The declining cost of sequencing library construction, sample multiplexing, and the advent of skim sequencing has reduced costs and enabled large-scale genetic and genomic experiments. Here, we present a simple skim sequencing and bioinformatics pipeline sufficient for various genotyping applications. Our low-depth skim sequencing method costs 21 USD per sample and provides an average of 144k reads. Our approach uses a double-stranded DNA sample to prepare libraries for genome sequencing. We demonstrate various uses for this strategy in maize, a complex and large (2.5 Gbp) genome. DNA from multiple pedigreed populations, including advanced backcrossed progenies, bi-parental populations, near-isogenic lines, and recombinant inbred lines, were used to map loci, detect donor introgressions, and determine introgression haplotypes. Read counts at known polymorphic positions detected donor genotypes even when derived from parents of unknown origin and could localize mutations of phenotypic impact via bulked segregant analysis. Remarkably, the small amount of sequencing data produced were sufficient to identify the haplotypes of introgressions of unknown origin from by comparison to known genotypes. Correct haplotype identification enabled more accurate allele frequencies to be calculated when mapping loci. This is of exceptional value in maize, where a rich collection of mutants from the 20th century are of unknown pedigree. One sentence summaryEconomical and efficient whole genome ultra-low pass sequencing of DNA samples for numerous genetic and genomic applications.

The Drosophila eye has been an important model to understand principles of differentiation, proliferation, apoptosis and tissue morphogenesis. However, a single cell RNA sequence resource that captures gene expression dynamics from the initiation of differentiation to the specification of different cell types in the larval eye disc is lacking. Here, we report transcriptomic data from 13,000 cells that cover six developmental stages of the larval eye. Our data show cell clusters that correspond to all major cell types present in the eye disc ranging from the initiation of the morphogenetic furrow to the differentiation of each photoreceptor cell type as well as early cone cells. We identify dozens of cell type-specific genes whose function in different aspects of eye development have not been reported. These single cell data will greatly aid research groups studying different aspects of early eye development and will facilitate a deeper understanding of the larval eye as a model system.

Resistance to starvation is a classic complex trait where genetic and environmental variables can greatly modify an animals ability to survive without nutrients. Genetic analyses in Drosophila have shown that starvation resistance is highly polygenic with different genetic architectures in different mapping populations. In this study we sought to dissect the genetic basis of starvation resistance among a set of mitonuclear genotypes carrying different mtDNAs paired with specific nuclear genomes from the Drosophila Genetic Reference Panel (DGRP). We focused on differences between one of the most sensitive strains (DGRP-765) and a strain with more moderate resistance (DGRP-315) whose starvation phenotypes appeared to be modified by alternative mtDNAs. Using complementary pooled-sequencing and forward genetic mapping approaches, we identified regions of chromosomes 2L, 3L and 3R contributing to starvation sensitivity and localize a major effect locus modifying starvation resistance to the coding region of phospholipase iPLA2-VIA. These analyses further confirm that the alternative mtDNAs had little influence on variation in starvation resistance between the genotypes studied. The sensitive line shows a starvation-dependent depletion of glucose and glycogen that is modified by hemi- and heterozygosity in the iPLA2-VIA region. These findings contribute to our understanding of the complex genetic relationship between resistance to starvation stress and nutrient metabolism.

Germ cell development and gamete production in animals require small RNA pathways. While studies indicate that microRNAs (miRNAs) are necessary for normal sperm production and function, the specific roles for individual miRNAs are largely unknown. Here, we use small RNA sequencing of dissected gonads and functional analysis of new loss of function alleles to identify functions for miRNAs in the control of fecundity and sperm production in Caenorhabditis elegans males and hermaphrodites. We describe a set of 29 male gonad-enriched miRNAs and identify a set of 3 individual miRNAs (mir-58.1, mir-83, and mir-235) and a miRNA cluster (mir-4807-4810.1) that are required for optimal sperm production at 20{degrees}C and 5 additional miRNAs (mir-49, mir-57, mir-261, and mir-357/358) that are required for sperm production at 25{degrees}C. We observed defects in meiotic progression in mir-58.1, mir-83, mir-235, and mir-4807-4810.1 mutants that may contribute to the reduced number of sperm. Further, analysis of multiple mutants of these miRNAs suggested complex genetic interactions between these miRNAs for sperm production. This study provides insights on the regulatory roles of miRNAs that promote optimal sperm production and fecundity in males and hermaphrodites. Article SummaryMicroRNAs are small non-coding RNAs that are required for the normal production of sperm but the roles of individual microRNAs in the process of spermatogenesis are not well understood. Here, we use the nematode Caenorhabditis elegans to identify microRNAs that are enriched in the male gonad to identify specific microRNAs that regulate male fertility. We generated new loss of function mutants for functional analysis to identify a set of microRNAs that are necessary for optimal fertility and fecundity in males.

Penstemon is the most speciose flowering plant genus endemic to North America. Penstemon species diverse morphology and adaptation to various environments have made them a valuable model system for studying evolution, but the absence of publicly available reference genomes limits possible research directions. Here we report the first reference genome assembly and annotation for Penstemon davidsonii. Using PacBio long-read sequencing and Hi-C scaffolding technology, we constructed a de novo reference genome of 437,568,744 bases, with a contig N50 of 40 Mb and L50 of 5. The annotation includes 18,199 gene models, and both the genome and transcriptome assembly contain over 95% complete eudicot BUSCOs. This genome assembly will serve as a valuable reference for studying the evolutionary history and genetic diversity of the Penstemon genus.

Drosophila melanogaster and Caenorhabditis elegans are well-used invertebrate models for studying the innate immune system. The organisms are susceptible to bacterial pathogens that include Pseudomonas species, (entomophilia - Drosophila) or (aeruginosa - Caenorhabditis), E. faecalis and P. rettgeri, which are or are related to human pathogens. Further, the consequences of exposure to these pathogens, in terms of organismal survival, are roughly equivalent when compared. That is, worms and flies are more susceptible to infection by Pseudomonas than E. faecalis, whereas organismal survival on E. faecalis and P. rettgeri are roughly the same in both. To better understand how these organisms are coordinating their responses to these bacterial pathogens we examined transcriptomes in infected animals. We grouped our analysis based on protein orthology. Of the 3611 pairs analyzed, we found genes whose responses were conserved across the different species at a higher than expected rate for two of the three pathogens. Interestingly within the animals, genes with 1:1 orthologs between species behaved differently. Such genes were more likely to be expressed in D. melanogaster, and less likely to be expressed in C. elegans. From this analysis we found that the gene nucleobindin (nucb-1/NUCB1 in C. elegans and D. melanogaster, respectively) was upregulated in both species in response to Gram negative bacteria. We used RNAi to knock down nucb-1 and found the treated animals were more susceptible to infection by the Gram negative pathogen P. rettgeri than controls. These results provide insight into some of the conserved mechanisms of pathogen defense, but also suggest that these divergent organisms have evolved specific means to orchestrate the defense against pathogens. Article SummaryWe analyzed transcriptomic data from C. elegans and D. melanogaster to compare the expression of orthologous pairs of genes in response to bacterial pathogens. Our results indicated that only a handful of genes that are orthologous between species are differentially expressed in response to pathogens, but that the pattern of expression was different when comparing one-to-one orthologs versus those that are restricted to one of the two organisms. These results suggest that, although broad patterns of susceptibility to bacterial pathogens are conserved, the regulatory framework by which the organisms fight pathogens is less well conserved. Further our results suggest a more complete analysis of the evolutionary changes in organismal responses to pathogens is required.

The composition of the gut microbiome is impacted by a complex array of factors, from nutrient composition and availability, to physical factors like temperature, pH, and flow rate, as well as interactions among the members of the microbial community. Many of these factors are affected by the host, raising the question of how host genetic variation impacts microbiome composition. Though human studies confirm this type of role for host genetics, its overall importance is still a subject of debate and remains difficult to study. The mouse model, by allowing the strict control of genetics, nutrition, and other environmental factors, has provided an excellent opportunity to extend this work, and the Diversity Outbred (DO) mice in particular present a chance to pinpoint host genetic variants that influence microbiome composition at different levels of generality. Here, we apply 16S rRNA gene sequencing to fecal samples of 247 DO male mice to estimate heritability and perform taxon-specific QTL mapping of microbial relative abundances revealing an increasingly heterogeneous picture of host function and microbial taxa at the host-microbiome interface. We present the first report of significant heritability of phylum Tenericutes in mice, and find novel QTL-spanning genes involved in antibacterial pathways, immune and inflammatory disease, and lipid metabolism.