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[
International Worm Meeting,
2021]
The genome size variation is an "old" question in evolutionary biology. However, its causes and consequences are still a debate. There are various intraspecies models to study genome size variations, which usually hold very limited size change between pairs. As a result, sophisticated and pricy detection methods are required to monitor the change of genome size. And the impact of large size variation can not be tested. The inter-species models, however, could have significant genome size variation. One close sister pair among Caenorhabditis clade, the C. briggsae and C. nigoni, has around 30% genome size difference. They can cross with each other and produce fertile females, and can be used to study genome size variation and hybrid incompatibility. One major disadvantage of inter-species model is that, the F1 male is either dead for sterile, which hinder the monitoring of further offspring by F1 crossing. We generated a lot of introgression strains with C. nigoni genome background with a small fragment from C. briggsae genome. By incorporating different C. briggsae X chromosome fragments into C. nigoni background, we are able to produce a homozygotic introgression strain (ZZY10253), which could mate with C. briggsae male and produce both fertile F1 females and males. By breeding the F1 worms with 10 x 10 crossing, we can monitor the competition of the two haplotypes, which has 30% size difference, by checking the ratio of two haplotypes using NGS sequencing. We have sequenced some F7 and F20 lines. And the long haplotype, the C. nigoni haplotype, has become dominant (78%) in F7 populations, especially in the X chromosome (92%). With this speed, we expected to see a 100% recovery of ZZY10253 genotype after F15. But to our surprise, the F20 lines still maintained at least 8% of C. briggsae haplotype, and all these lines (n=8) have two autosomes remained. These remained autosomes may reflect an interaction between the X introgression fragment and autosomes, which grant these individuals some advantage in growing.
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Zhao, Zhongying, Li, Runsheng, Young, Amanda, Zhang, Zhihong, Ren, Xiaoliang, Hsieh, Chia-Ling
[
International Worm Meeting,
2015]
Next-Generation Sequencing permits rapid acquisition of high-throughput DNA sequences, but its reads are relatively short in length, limiting their use in genome-based studies. Illumina has recently released a technology called Synthetic Long-Read Sequencing that can produce reads of unusual length, i.e., predominately around 10 Kb. However, a systematic assessment of their use in genome finishing and assembly is still lacking, especially in resolving the repetitive sequences. We evaluate the promise and deficiency of the long reads in these aspects using C. elegans genome. First, the reads are highly accurate and capable of recovering most types of repetitive sequences. However, the presence of tandem repetitive sequences that extend over certain length prevents pre-assembly of the long reads inside this genomic region. Second, the reads can reliably recover the missing but not the extra sequences in C. elegans genome. 24X of the long reads allow the recovery of at least 40 Kb of missing genomic DNAs that are located inside or outside coding region. Finally, an N50 size of at least 86 Kb can be achieved for the contigs that are de novo assembled with the 24X reads. However, substantial mis-assembly errors are observed which are caused by either mis-assembled long read or flanking repetitive sequences, highlighting a need for novel assembly algorithm to accommodate the long reads in de novo genome assembly. The long reads are expected to be useful in generating a "finished-grade" genome of other nematode species if coupled with independent data such as those from PacBio or mate-pair sequencing or repairing the existing nematode draft genomes on its own.
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[
International Worm Meeting,
2017]
Differential expression of orthologous genes could be a product of divergence in cis-regulatory elements and/or trans-factors. Analysis of transcriptomes in F1 hybrids between closely related species provides an opportunity for systematically dissecting the roles of cis-elements and the trans-factors in controlling allele-specific expression. This analysis could also provide insight into the mechanism controlling genome stability, for example, through transposon silencing. The F1 hybrid transcriptomes have been analyzed in most model organisms, but never been examined in nematode species. Here we perform transcriptome analyses in the F1 female hybrids from reciprocal crosses between C. briggsae and C. nigoni with their parental hermaphrodites/females as a control. The reciprocal transcriptomes demonstrate a nearly perfect correlation between each other, indicating few genomic imprinting events in the parents. Approximately 8,000 orthologous pairs have at least 10 sequencing reads which are used in the subsequent analysis. Over 5,000 of them demonstrate differential allelic expressions in the hybrids, which mirror their expression divergences in their respective parents, indicating that cis-regulation plays a major role in controlling expression of these pairs in the hybrids. Roughly another 800 pairs show a significant averaging effect of their parental expression divergences in the hybrids, supporting the allelic expression in the hybrids is predominately regulated by trans-factors. The remaining 2,000 pairs demonstrate conserved expression in both parents and their hybrids. Unlike the F1 hybrids in Drosophila species in which a sharp increase in transposon expression is frequently observed, we barely see any significant increase in the expression of the mobile elements, suggesting compatible endogenous RNAi pathways between the two species in transposon silencing. Taken together, we demonstrate a lack of parent-of-origin differential allelic expression in female reciprocal F1 hybrids between C. briggsae and C. nigoni, in which allele-specific expressions of orthologous pairs are mostly controlled by cis-regulatory elements. Transposon-silencing mechanisms appear to be conserved between the two species.
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[
International C. elegans Meeting,
1997]
Lithium (Li) has long been known to have teratogenic effects on the development of many organisms, including sea urchin, Xenopus and Dictyostelium. In Li-treated Xenopus embryos, ventral blastmeres are respecified to develop into dorsal structures, leading to dorsalized embryos lacking ventral mesodermal tissues. In Dictyostelium, Li alters the fate of prespore cells to become prestalk cells instead. Besides teratogenic effects, Li is also known to be a most effective treatment of manic-depressive illness.!@Although several models have been proposed to explain Li action, the molecular mechanism has remained unclear. Recently, GSK (glycogen synthase kinase)-3b was proposed to be a target of Li, suggesting that Li affects the wnt signaling pathway. To understand the mechanism of Li action, I first examined the effect of Li on C. elegans embryogenesis. I inoculated N2 animals at the late L4 stage onto NG plates containing 20 mM LiCl, incubated them at 20 oC. The number of eggs produced by treated animals was reduced to about half of the untreated control. Although cell division seemed to proceed, no embryos hatched on Li plates. Treated-embryos developed to produce gut granules, but did not execute normal morphogenesis at later embryonic stages. To identify genes involved in the action of Li, I have begun to screen for Li-resistant mutants that propagated on Li-containing medium. Several mutants were isolated, and one of them was mapped on the left side of
unc-42 on chromosome V. On Li plates, the hatching rate of mutant eggs cross-fertilized by wild-type males was essentially the same as that for self-fertilized mutant eggs. On the contrary, no wild-type eggs cross-fertilized by mutant males hatched on Li-containing plates. So, this mutation appeared to be maternal. Further genetic analyses of the mutants and the observation on the cellular phenotype of Li-treated embryos are underway.
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[
International C. elegans Meeting,
2001]
Little is known about how ions permeate the C. elegans gut and cuticle. How various ions influence development and behavior is also not understood. One ion with considerable impact in human biology is Li + , which modulates bipolar disorder by an unknown mechanism. In particular, the targets of lithium that cause side effects remain to be identified. We are using C. elegans to genetically identify targets of lithium and to elaborate on mechanisms of transport, which may have an impact on human health. Li + has dosage-dependent effects on C. elegans embryos. When L4 animals mature on NGM plates with Li + added to the final concentrations of 10mM-20mM, they produce embryos that are unable to hatch. We demonstrated that this failure to hatch is due to defects in cytokinesis that result in multi-nucleated embryos and symmetrically partitioned cells. Li + also has a dosage-dependent effect on larval development. When adult hermaphrodites are permitted to lay embryos on 10mM-20mM Li + NGM plates, the resulting offspring experience a developmental delay proportional to the concentration of Li + . The offspring become progressively paralyzed as they reach adulthood. We demonstrated earlier that there is a delay in the entry into the S phase of the cell cycle larval stages. To learn more about the biology of Li + sensitivity, we screened for Li + resistant mutants. We developed three screens that took advantage of the embryonic arrest and the larval delay caused by Li + . We isolated one mutant
bz71 , in a screen of 44,000 haploid genomes, that is resistant to both the larval and embryonic blocks of 16mM Li + .
bz71 is dominant. Using classical mapping techniques, we positioned it on LGIII between
unc-32 and
dpy-18 . We are currently using SNP strategy to obtain a higher resolution map and we hope to report on the identification and cloning of this locus.
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Li, Runsheng, Xie, Dongying, Ding, Qiutao, Ren, Xiaoliang, Zhao, Zhongying, Bi, Yu
[
International Worm Meeting,
2019]
High throughput RNA-seq using cDNA has played a key role in delineating transcriptome complexity, including alternative transcription initiation, splicing, polyadenylation and base modification. However, the reads derived from current RNA-seq technologies are usually short and deprived of information on modification during reverse transcription, compromising their potential in defining transcriptome complexity. Here we applied direct RNA sequencing with ultra-long reads from Oxford Nanopore Technologies (ONT) to study the transcriptome complexity in C. elegans. We sequenced native poly-A tailed mRNAs by generating approximately six million reads from embryo, L1 larvae and young adult animals, with average read lengths ranging from 900 to 1,100 bps across stages. Around half of the reads represent full-length transcripts judged by the presence of a splicing-leader or their full coverage of an existing transcript. To take advantage of the full-length transcripts in defining transcriptome complexity, we devised a novel algorithm to predict and quantify the isoforms using read's mapping tracks rather than established intron/exon structures. We recovered at least 26,000 out of the existing 33,500 isoforms, and identified roughly 56,000 novel ones. Intriguingly, stage-specific expression at the level of gene and isoform demonstrates little correlation. Finally, we observed an elevated level of modification in all bases in the coding region than the UTR. Taken together, the native long reads by ONT are expected to deliver new insights into RNA processing and modification and their underlying biology.
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[
East Coast Worm Meeting,
2004]
Targeting Induced Local Lesions In Genomes, or TILLING R , is a novel high throughput technology for reverse genetics. TILLING uses chemical mutagenesis to yield a traditional allelic series of point mutations for virtually all genes. TILLING is of particular value for essential genes where sublethal alleles are required for phenotypic analysis. TILLING has become an established technique on many model organisms, including C. Elegans, Arabidopsis, Zebrafish, Maize, Rice, and others. A variation on TILLING is called Ecotilling, in which high throughput SNP discovery is performed by locating natural variations throughout the genome. LI-COR Biosciences is a manufacturer of DNA Analysis Instrumentation for high throughput TILLING and Ecotilling, as well as for DNA Sequencing, AFLP R , and Microsatellite analysis. LI-COR also manufactures instrumentation for infrared fluorescent protein imaging, offering superior quantification over standard chemiluminescence. Stop by our booth to pick up the latest literature and publications on our products. TILLING is a registered trademark of Anawah, Inc. AFLP is a registered trademark of KeyGene, N.V.
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Xie, Dongying, Bi, Yu, Li, Runsheng, Zhao, Zhongying, Yan, Cheung, Shao, Jiaofang, Ren, Xiaoliang
[
International Worm Meeting,
2015]
Genetic basis of hybrid incompatibility (HI), a collective term of compromised fitness in hybrid, including sterility and lethality, has been intensively studied in Drosophila species, but remains poorly understood in other species, especially in nematodes. Recent discovery of a C. briggsae sister species, C. nigoni, has opened up the possibility of dissecting the genetic basis of HI in nematode species. We have generated 96 chromosomally integrated GFP markers in the C. briggsae genome and mapped them into the defined locations by PCR and Next-Generation Sequencing (NGS). Aided by the marker, we backcrossed the GFP-associated C. briggsae genomic fragments into C. nigoni for at least 15 generations and produced 111 independent introgression lines. The introgression fragments cover most of the C. briggsae genome. We finally dissected the patterns of HI by scoring the embryonic lethality, larval arrest, sex ratio and male sterility for each introgression line, through which we identified pervasive HI loci and produced a genome-wide landscape of HI between the two nematode species, the first of its type for any non-Drosophila species.To further understand the genetic basis of HI between the two species, we focused on the molecular understanding of two introgression fragments that produce male sterility but are located on different parts of X chromosome. To this end, we performed RNA-seq for both sterile and parental males and quantified the reads using C. briggsae genome as a reference. A total of 957 genes were found differentially expressed (Fold Change>1.5, FDR<0.05) in both hybrids compared with both parental species with most genes showing down-regulation. Surprisingly, 364 out of 957 genes are those enriched during spermatogenesis, suggesting that preferential down-regulation of spermatogenesis genes caused by the introgression may at least partially to be blamed for the observed sterility. More details on the differentially expressed genes will be presented in the meeting.
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[
International Worm Meeting,
2019]
Insulin-like signaling pathway plays diverse roles in metabolism, growth, longevity, development, and behaviors in the animal kingdom. C. elegans genome encodes 40 insulin-like genes (Duret et al., 1998; Pierce et al., 2001; Li et al., 2003; Li and Kim, 2008), which regulate development and behaviors. Also, C. elegans releases small molecules ascarosides, which control dauer formation, sex-specific and social behaviors. We and others previously showed that C. elegans hermaphrodite exhibits acute avoidance behavior to ascr#3 (asc-?C9, C9) exposure via the ascr#3-sensing ADL neurons (Jang et al., 2012). Moreover, we also showed that feeding state affects ascr#3 avoidance through DAF-2 insulin-like receptor (Ryu et al., 2018). To further investigate role of ILPs in ascr#3 avoidance, we examined contribution of INS-1 to ascr#3 avoidance. We first found that
ins-1 mutant animals decrease ascr#3 avoidance behaviors in well-fed condition. We are currently identifying where and how INS-1 acts to mediate ascr#3 avoidance. Additionally, we are testing whether energy-sensing AMPK signaling affects ascr#3 avoidance. Key words: ILPs, Pheromone Avoidance, Starvation. AMPK signaling
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[
International C. elegans Meeting,
1999]
The egg-laying motorneurons release multiple neurotransmitters, each of which appears to control a specific aspect of egg-laying behavior. Under favorable conditions, wild-type worms fluctuate between alternative behavioral states for egg-laying: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Switching between states, as well as egg-laying within the active state, are stochastic, Poisson-like processes. We showed previously that serotonin, released primarily from the HSNs, facilitates the switch from the inactive to the active state, while acetylcholine, released from both the HSNs and VCs, promotes egg-laying within the active state. Li and Chalfie have shown that both the HSNs and VCs contain one or more FMRFamide-related peptides, and provided pharmacological evidence that these neuropeptides may control of egg-laying behavior. To investigate the roles of these peptides in more detail, we have analyzed the egg-laying behavior of deletion mutants (kindly provided by Chris Li) that are defective in production of specific FaRP precursors. We have found that one of these mutants, defective in the gene
flp-1, have dramatically lengthened inactive phases, but completely normal egg-laying within the active phase. Moreover, although these mutants are stimulated to lay eggs by serotonin with approximately normal dose responses, their rate and pattern or egg-laying on serotonin are abnormal. Thus, we hypothesize that the FLP-1 peptides and serotonin act in parallel to affect the same step in egg-laying behavior--inducing the switch between the inactive and active phases. Experiments to investigate the cellular and molecular mechanisms for the FLP-1 peptides' affects on egg-laying will be described. Thanks to Chris Li for discussions and for providing strains.