Andersen, Erik et al. (2021) International Worm Meeting "The Caenorhabditis Natural Diversity Resource: expanded and enhanced"

Andersen, Erik, Tanny, Robyn, Evans, Kathryn

[International Worm Meeting, 2021]

Natural diversity offers a treasure trove of variants (or mutations) that can be used to understand quantitative traits and evolutionary processes. The Caenorhabditis elegans Natural Diversity Resource (CeNDR) has archived and disseminated wild strains, whole-genome sequenced those strains, and released variant data and effect predictions to the community. Also, CeNDR facilitates genome-wide association (GWA) mappings to connect natural differences in quantitative traits to variants and molecular mechanisms. Since 2016, CeNDR has shipped more than 4,000 strains and enabled over 3,000 GWA mappings for the community. We continue to archive and sequence new wild strains identified by collaborators and citizen scientists, offering more than 1,300 genetically unique strains. In addition to these new strains, we updated CeNDR to make it more user-friendly, created a broad-sense heritability calculator, modernized the variant browser and variant effect predictions, and created a powerful new GWA mapping portal. These additions and updates make CeNDR more accessible and able to facilitate natural variation studies in laboratories often focused on the laboratory-adapted N2 strain. We present the evolution of CeNDR into the Caenorhabditis Natural Diversity Resource (CaeNDR) where we have incorporated over 1,600 C. briggsae and 700 C. tropicalis wild strains and genome sequences. The same browsers and tools created for C. elegans are available for these species. CaeNDR will expand comparative genomics across the three selfing Caenorhabditis species and enable new approaches to quantitative genetics and population genomics.

Crombie, Timothy et al. (2021) International Worm Meeting "Direct estimate of the distribution of fitness effects (DFE) of spontaneous mutations in C. elegans"

Johnson, Lindsay, Andersen, Erik, Saxena, Ayush, Baer, Charles, Tanny, Robyn, Crombie, Timothy, Ponciano, Jose Miguel, Rajaei, Moein

[International Worm Meeting, 2021]

The distribution of fitness effects (DFE) of new mutations is a fundamental parameter in population genetics, and has practical application in the context of modeling the genetic basis of complex heritable disease in humans. However, the DFE is very difficult to estimate empirically. At present, nearly all estimates of the DFE rely on either statistical inference from the standing site-frequency spectrum or from laboratory estimates of fitness in mutation accumulation (MA) lines, with no reference to the underlying specific mutations. We report results from a new method to estimate the DFE from competitive fitness data from a set of recombinant inbred lines (RILs) derived from a cross between two C. elegans MA lines, combined with whole-genome sequence data from the set of >500 RILs and the parental lines, as well as from ~40 fully-sequenced MA lines. Of five distributions fit to the data, all produce similar results, but the best fit is to a normal distribution nearly centered at 0, with almost no weight beyond +/- 2%. Averaged over two fitness assays, nearly ten years apart, the average fitness effect of a new mutation is about -0.2%. These results are in strong contrast with estimates of the average mutational effect inferred from MA line fitness data not informed by sequence data, which are much larger. We suspect that a few mutations of large effect may have been lost during the inbreeding phase of the construction of the RILs.

Zamanian, M et al. (2017) International Worm Meeting "Evidence of small RNA involvement in nematode benzimidazole resistance."

Lee, Junho, Zdraljevic, Stefan, Andersen, Erik, Rodriguez, Briana, Lee, Daehan, Tanny, Robyn, Cook, Daniel, Brady, Shannon, Zamanian, M

[International Worm Meeting, 2017]

Parasitic nematodes impose a debilitating health and economic burden across much of the world. The morbidity and mortality inflicted by these pathogens are partly curtailed by mass drug administration programs that depend on the continued efficacy of a limited portfolio of anthelmintic drugs. Benzimidazoles are WHO-designated "Essential Medicines" and an indispensable component of this limited arsenal. Nematode resistance to benzimidazole chemotherapy threatens parasite control efforts in both human and veterinary medicine. Despite this threat, the genetic landscape of potential resistance mechanisms to thiscritical drug class remains largely unexplored. There is an urgent and recognized need to identify molecular mechanisms and genetic markers that cause benzimidazole resistance. In order to identify conserved nematode drug responses, we exploit natural variation in two model roundworms, Caenorhabditis elegans and Caenorhabditis briggsae, to discover quantitative trait loci (QTL) that control benzimidazole sensitivity. Surprisingly, we found that resistance to benzimidazoles mapped to syntenic piRNA-enriched regions of the genome with few protein-coding genes in both Caenorhabditis species. We used near isogenic lines (NILs) to narrow the major-effect benzimidazole QTL in C. elegans to a smaller region of the genome and demonstrate that the benzimidazole-resistance phenotype results from piRNA variation that is dependent on the function of the piRNA-associated argonaute prg-1. We identified candidate piRNAs causal to the resistance phenotype and putative genes targeted for silencing by downstream 22G RNAs. Our results indicate that small RNAs require consideration in drug resistance mechanisms in nematodes, because the piRNA pathway and related small RNA pathways are conserved in many medically and agriculturally important parasitic nematodes. Importantly, this resistance mechanism could be mediated through the heritable transgenerational effects of small RNAs. This finding has significant implications for parasite control and the management of drug resistance in other phyla and systems.

Webster, Amy et al. (2021) International Worm Meeting "Natural genetic variation in irld genes modifies insulin signaling to influence starvation resistance"

Chen, Jingxian, Powell, Maya, Webster, Amy, Chitrakar, Rojin, Evans, Kathryn, Andersen, Erik, Stevens, Lewis, Antoshechkin, Igor, Fisher, Kinsey, Baugh, Ryan, Tanny, Robyn

[International Worm Meeting, 2021]

The genetic basis of natural variation in starvation resistance is not well understood though it is a fundamental, biomedically important trait. We developed a population selection and sequencing approach (MIP-seq) to measure starvation resistance for a large number of wild C. elegans strains in a single culture. We identified three quantitative trait loci (QTL) affecting starvation resistance. These QTL overlap with hyper-divergent regions and contain multiple members of several large gene families involved in environmental interactions. In particular, we identified 16 members of the insulin/EGF receptor-like domain (irld) family with variants within starvation resistance QTL. We generated and assayed loss-of-function mutations for four irld family members, all of which increased starvation resistance. We show that the transcription factor daf-16/FoxO, a critical effector of insulin/insulin-like growth factor signaling (IIS) known to promote starvation resistance, is required for increased resistance of irld-39; irld-52 mutants, that these mutants affect DAF-16 target gene expression, and that the IIS receptor daf-2/InsR is epistatic to these irld genes. We propose that IRLD proteins bind insulin-like peptides (ILPs) to modify signaling in the sensory nervous system thereby affecting organismal physiology. This work demonstrates the efficacy of using population sequencing to investigate natural variation of a complex trait, and it identifies irld genes that regulate IIS and starvation resistance. Furthermore, it shows that variation in a rapidly evolving large gene family modifies activity of a deeply conserved signaling pathway to affect a fitness-proximal trait.

Moya, Nicolas D. et al. (2021) International Worm Meeting "Improved reference genomes for Caenorhabditis briggsae"

Chitrakar, Rojin, Stevens, Lewis, Baugh, L. Ryan, Moya, Nicolas D., Walhout, Marian, Tanny, Robyn E., Dekker, Job, Na, Huimin, Andersen, Erik C.

[International Worm Meeting, 2021]

Decades of research have led to the development of comprehensive genome resources that have been essential to study the Caenorhabditis elegans species. In parallel, the emergence of Caenorhabditis briggsae as a model system has been useful to make interspecies comparisons. Despite the importance of C. briggsae as a model, its genome resources have not been developed to the same extent as C. elegans. The current genome of C. briggsae reference strain AF16 contains thousands of unresolved gaps and numerous mis-assemblies. Because of these issues, C. briggsae gene models remain incomplete and have numerous structural errors in protein-coding genes. We sought to exploit the latest sequencing technologies and computational tools to provide the highest quality C. briggsae genome resources to date. First, we generated high-quality genome assemblies for two strains of C. briggsae: QX1410 (a "tropical" strain isolated in Saint Lucia that is closely related to AF16) and VX34 (a divergent strain isolated in China). These genome assemblies incorporate high coverage Oxford Nanopore PromethION long reads and chromosome conformation capture (Hi-C) data. Second, we genotyped 99 recombinant inbred lines generated from reciprocal crosses between QX1410 and VX34. Using these data, we produced a high-quality recombination map that validated the placement of scaffolds after genome assembly. Third, we sequenced the transcriptomes of each strain to high coverage using Pacific Biosciences SMRT and Illumina platforms. We developed a computational pipeline that leverages long and short RNA reads to generate a genome annotation for each strain. These new genome annotations have improved accuracy and completeness relative to the AF16 genome. Fourth, our research group currently maintains over 1,600 C. briggsae wild strains, comprising the largest collection worldwide. We sequenced the genomes of this entire collection to high coverage using the Illumina platform. We mapped the sequences of all wild strains to the QX1410 genome to call single nucleotide variants across the entire population. These high-quality genome resources will facilitate new avenues of research, including quantitative and population genetic studies of C. briggsae, and enable informative comparisons with C. elegans.

Rajaei, Moein et al. (2021) International Worm Meeting "Mutability of mononucleotide repeats explains the discrepancy between lab-accumulated mutations and the natural allele frequency spectrum of C. elegans"

Rajaei, Moein, Andersen, Erik C., Baer, Charles F., Crombie, Timothy A., Tanny, Robyn E., Johnson, Lindsay M., Snyder, Michael C., Saxena, Ayush Shekhar, Joyner-Matos, Joanna

[International Worm Meeting, 2021]

Mutation is the fuel of evolution, and is of fundamental importance in evolutionary biology. A usual and efficient way to estimate the properties of spontaneous mutation divorced from the influence of natural selection is by means of mutation accumulation (MA) experiments, in which the efficacy of selection is minimized. However, MA experiments potentially come with their own biases. Previous studies have consistently shown a discrepancy between the mutation spectrum observed in MA lines and the site frequency spectrum (SFS) of wild isolates. By applying MA experiments and whole genome sequencing to three strains of C. elegans, (N2, PB306, and an N2-derived strain carrying a defective allele at the mev-1 gene), we investigated the property of spontaneous mutations in different part of the genome. We find that the mutational properties of mononucleotide repeats differ in both rate and spectrum from non-mononucleotide sequences, both for base-substitutions and insertion/deletion (indel) mutations. Comparison of the MA spectrum to that of segregating "private" alleles (which have presumably arisen recently as new mutations) reveals that the spectra at non-mononucleotides are similar between MA lines and wild isolates, whereas the mononucleotide spectra are very different, both for base-substitutions and indels. In addition, we compared the mutational properties of highly divergent regions of the C. elegans genome to those of weakly diverged regions. Our preliminary analysis suggests that the mutation rate is slightly higher in divergent regions, but the difference in mutation rate is not nearly large enough to explain the difference in nucleotide diversity.

Crombie, Tim et al. (2021) International Worm Meeting "Repeated Sampling of Caenorhabditis elegans Across the Hawaiian Islands Reveals Spatiotemporal Patterns of Genetic Diversity"

Stevens, Lewis, Buchanan, Claire, Andersen, Erik, Stinson, Loraina, Dilks, Clayton, Tanny, Robyn, Lu, Dan, Zhang, Gaotian, Evans, Kathryn, Zdraljevic, Stefan, Crombie, Tim, Lee, Daehan, Roberto, Nicole, Wang, Ye, Cook, Daniel

[International Worm Meeting, 2021]

Caenorhabditis elegans isolated from the Hawaiian Islands are known to harbor a high degree of genetic diversity relative to non-Hawaiian isolates. It was recently suggested that Hawaiian C. elegans can be partitioned into at least four genetically distinct groups. An analysis of geospatial environmental data further suggested that the genetic groups might associate with environmental parameters such as elevation and temperature, although the sample size for that study was small (n = 43 isolates). To better characterize the niche and genetic diversity of Hawaiian C. elegans and further define the associations of genetic groups with environmental parameters, we sampled different substrates and niches across the Hawaiian Islands six times over a three-year period. In total, we isolated 7,107 nematodes from 2,400 of 4,506 substrate samples (53% success rate). Among the nematodes we isolated, we identified five Caenorhabditis species, including 499 C. elegans, 377 C. briggsae, and 55 C. tropicalis isolates. We measured several environmental parameters at each sampling site and combined them with environmental parameters from geospatial databases to reveal that C. elegans is typically found in cooler and relatively drier climates at higher elevation than the other two selfing Caenorhabditis species. We isolated C. elegans most frequently from montane-alpine mesic forest habitat dominated by plant species native to the Hawaiian Islands. When possible, we cryopreserved C. elegans isolates and sequenced their genomes. To date, including Hawaiian isolates from collaborators, we have sequenced the genomes of 505 Hawaiian C. elegans isolates. With these data, we grouped the isolates into 163 isotypes (strains belonging to a single isotype have >0.9997 genome-wide concordance). We found that some of the isotypes were collected from the same locations over the three-year sampling period, and most of the collections of the same isotype were found within 500 meters of each other. Principal component analysis (PCA) of genetic variation revealed that the 163 isotypes fall into seven genetically distinct groups, three more than previously found on the islands with a smaller sample. Taken together, our findings begin to outline the spatiotemporal patterns of C. elegans genetic diversity on the Hawaiian Islands and raise new questions about evolutionary forces driving the genetic structure we have uncovered. For example, are these groups isolated by ecological or geographic distances, or perhaps both, and to what extent do reproductive incompatibilities contribute to the structure we have observed?

H Schwartz et al. (1999) International C. elegans Meeting "A screen for mutants defective in the specification of programmed cell death in the postdeirid lineage"

B Horvitz, H Schwartz

[International C. elegans Meeting, 1999]

During wild-type hermaphrodite development, 131 somatic cells undergo programmed cell death. While many genes involved in the execution of cell death have been identified, the mechanisms that control the commitment of specific cells to undergo programmed cell death are poorly understood. To date, mutations in three genes, ces-1 , -2 , and -3 ( ce ll death s pecification), have been found that affect specifically the deaths of particular cells. ces-1 and ces-2 have been cloned and shown to encode transcription factors. We intend to perform a genetic screen for ces genes involved in the deaths of the sisters of the PVD neurons in the postdeirid lineage. We chose this lineage because the deaths occur during postembryonic development, making the deaths easy to identify using Nomarski optics, and because reporters exist that seemed likely to be expressed in these cells if their deaths were prevented by a mutation affecting programmed cell death. In ced-3 mutants that appear to be completely defective in programmed cell death, roughly half of the "undead" PVD sisters contain dopamine, like their "aunt" the PDE cell, as seen by formaldehyde-induced fluorescence. We have found that GFP reporters for dopaminergic neurons are expressed in 60% of undead PVD sisters in mutants defective in programmed cell death. We are beginning a screen using cat-2 ::GFP (kindly provided by Robyn Lints) to identify mutants in which the PVD sisters survive.