Genetics最新文献

Gene regulation via Argonaute-bound small RNAs is a broadly conserved mechanism, present in all domains of life. The nematode Caenorhabditis elegans expresses several worm-specific Argonautes (WAGOs), which interact with the small RNAs known as 22G-RNAs. These WAGOs have roles in gene regulation, transposon-defence as well as in viral control and experimentally induced RNA interference. Despite many studies, direct relationships between WAGO targeting, as defined by 22G-RNA sequences, and mRNA abundance are not clear. Also, the effects of developmental stage and WAGO-interconnectivity have been under-studied thus far. We studied these aspects for two germline-expressed WAGO proteins, WAGO-1 and WAGO-3. We show that WAGO-1 mostly affects 22G-RNA expression in gravid adult worms, while WAGO-3 predominantly affects 22G-RNA expression in embryos. Furthermore, we detect a link between WAGO-3 and the maternal 26G-RNA pathway governed by the Argonaute protein ERGO-1, and between WAGO-1 and the paternal 26G-RNA pathway governed by the Argonautes ALG-3/4. We also demonstrate that, globally speaking, loss of WAGO-1 or WAGO-3 does not result in upregulation of their target mRNAs, as defined by 22G-RNA complementarity. Finally, metagene analysis of 22G-RNA profiles suggests loss of one WAGO protein leads to shifts in WAGO 22G-RNA binding. Overall, we conclude that WAGO-1 and WAGO-3 are developmentally dynamic, are embedded in distinct regulatory networks, and that potential silencing of individual mRNAs by these WAGO proteins is hard to assess by simple loss-of-function studies.

Genome-wide association studies (GWAS) are a foundational tool in human genetics research, however, challenges in stability and reproducibility of GWAS results are often noted. The main goals of this work are to describe, analyze, and provide tools for solving such reproducibility challenges in a popular component of GWAS literature: set-based (a) hypothesis testing and (b) effect size estimation studies. Common forms of (a) include rare variant or gene-based association studies, while (b) frequently occurs in polygenic score construction and fine-mapping studies. Specifically, we focus on how the set-based natures of (a) and (b) often fuel non-reproducible results due to seemingly innocuous differences in data processing pipelines that are rarely discussed. Such obstacles present enormous challenges for the robustness and reliability of GWAS findings. First, we describe the processing challenges both qualitatively and quantitively, casting the statistical models in a model misspecification framework. Second, we analytically calculate the differences in power and amounts of bias that can arise in (a) and (b), respectively, due to small, relatively under-appreciated choices in data cleaning. Third, we provide tools for quantifying and avoiding the data quality obstacles in GWAS. We validate our analytical calculations through a simulation study, and we demonstrate the aforementioned challenges empirically through analysis of a pancreatic cancer dataset. In our analysis, we demonstrate that top associations, such as between pancreatic cancer and ATM, can be entirely lost due to small differences in data preparation, underscoring the need to make data processing choices clear and explicit.

The fresh-market blackberry (Rubus subgenus Rubus) industry has expanded dramatically in the past two decades, driven in part by improved cultivars. Introgression of the primocane-fruiting (PF; annual flowering) trait into elite germplasm has enabled dual cropping in a single year, season extension, and cultivation in tropical and subtropical regions. Despite its economic performance, the genetic basis of PF is not well understood. It has been proposed that the PF trait is controlled by a major recessive locus, but its genomic location is unclear. Here, a genome-wide association study (GWAS) of 365 tetraploid blackberry genotypes identified a single genomic region on chromosome Ra03 (∼33 Mb) strongly associated with PF. Genetic linkage analysis in a biparental population confirmed that the same interval (32-35 Mb) was linked to the PF phenotype. Ten putative candidate genes were identified in this region. Allele mining using whole-genome resequencing of 17 genotypes highlighted two high-priority candidates: a CCCH-type zinc finger gene and a ubiquitin-specific protease gene. Use of an improved Rubus argutus 'Hillquist' genome annotation (v1.2) enabled refined variant interpretation, including identification of regulatory 3' UTR polymorphisms in the zinc finger homolog. Two diagnostic KASP markers (PF1 and PF2), designed from the most significant GWAS SNPs, predicted the PF phenotype with over 96% accuracy in a validation panel of 494 tetraploid blackberries from multiple breeding programs. Together, these results provide the first high-resolution mapping of the PF locus in blackberry, identify candidate genes for flowering regulation in Rubus, and deliver diagnostic markers that can be immediately deployed in breeding programs.

Signals and preferences are central to the evolutionary origins and maintenance of reproductive barriers between species. How such communication systems evolve is key to understanding speciation. One central question considers how signals and preferences coevolve as lineages diverge. Here, we study the rapidly speciating Laupala crickets, a system characterized by coordinated sexual signaling and acoustic barriers to gene flow between species. By mapping a new locus underlying female song preference as well as fine-mapping a locus for male pulse rate of the acoustic communication system, we examine the genetic architecture underlying signal-preference divergence. We document a pair of colocalizing pulse rate and preference QTL on linkage group 4, with peak locations less than 2 cM apart, offering strong evidence for genetic coupling. Intriguingly, this is the third pair of colocalizing male and female loci mapped in the Laupala genome. The cumulative effect size of the three pairs of colocalizing loci account for roughly half of the interspecific difference, suggesting that genetic coupling has contributed substantially to the evolution of this behavioral barrier in Laupala. Annotation of the QTL region identified numerous functionally relevant candidate genes, including acetylcholinesterase (AChE) and ryanodine receptor (RyR).

Cell adhesion molecules (CAMs) play important roles in neurons, contributing to nervous system development, synapse formation and plasticity. A subset of CAMs, Claudins, known for their roles at tight junctions, remains underexplored in neurons. Recent studies in Caenorhabditis elegans have begun to reveal neuronal functions of claudin-like proteins. However, a systematic analysis of their neuronal expression has not been performed. We conducted a transcriptional reporter screen for claudin-like genes in C. elegans and identified several candidates showing neuronal expression, highlighting possible roles for claudins in the nervous system. One candidate, clc-3, showed robust expression in head, tail, and ventral cord neurons, with no detectable expression in non-neuronal tissues. Functional analyses of clc-3 mutants revealed increased body-bend amplitudes and elevated evoked postsynaptic currents at the cholinergic neuromuscular synapses. Imaging and molecular interaction studies demonstrated that CLC-3 likely interacts with the actin-binding protein NAB-1 to regulate cholinergic transmission. Our findings identify CLC-3 as a neuronally expressed claudin that regulates motor system output, likely through synaptic vesicle organization and illustrates how changes in synaptic function affect animal behavior.

Dosage compensation (DC) in C. elegans utilizes a condensin complex that resembles mitotic condensins, but differs by one subunit, DPY-27. DPY-27 replaces SMC-4, one of the Structural Maintenance of Chromosome (SMC) proteins that is responsible for hydrolyzing ATP, required for condensation of DNA and other mitotic condensin functions. To understand if the ATPase function is required in DC, we first demonstrated that DPY-27 is capable of hydrolyzing ATP in vitro. Then, we used CRISPR/Cas9-mediated genome editing to generate an ATPase mutation in dpy-27. Although the mutant protein is expressed and it is incorporated into the condensin IDC complex, this mutation results in a loss of DC. Specifically, we found that without ATPase function, DPY-27 containing condensin IDC has reduced capacity to bind DNA, condense the X chromosomes, and facilitate H4K20me1 enrichment on the X-chromosomes. Our results suggest that condensin IDC, like mitotic condensins, uses ATP hydrolysis to perform its functions, making C. elegans DC a model for how activities attributed to mitotic condensins can be used to regulate gene expression.

Failure to negatively regulate the activity of the bacterial DnaA initiator protein leads to overinitiation. The resultant elevated number of closely spaced replication forks increases the likelihood of lethal double strand DNA breaks when these forks encounter single stranded DNA gaps formed as an intermediate during excision repair. The principal mechanism underlying the regulation of DnaA relies on the ability of the Hda-β sliding clamp complex to stimulate DnaA's intrinsic ATPase activity to convert active DnaA-ATP into less active DnaA-ADP. Although an x-ray crystal structure of the Hda-β clamp complex was described (PDB: 5X06), it represents an inactive conformation. Using genetic and biochemical approaches, we identified two loop domains of the β clamp required for Hda function, but the enigma is that these loops do not interact with Hda in the crystal structure. Their possible roles in supporting Hda function are discussed.

Sperm display remarkable diversity in size and morphology, especially in insects. Sperm length varies by several hundred-fold within Drosophila alone. In the model species Drosophila melanogaster, sperm are approximately 1.7 times longer than those of its close relatives D. simulans and D. mauritiana. However, the developmental mechanisms underlying such rapid divergence in sperm traits remain poorly understood. Somatic hub and cyst cells are essential for the maintenance of germline stem cells in Drosophila spermatogenesis, yet their contribution to cell growth later in spermatogenesis remains unclear. Here, we used primordial germ cell (PGC) xenotransplantation to determine whether sperm length regulation is cell-autonomous or influenced by the somatic environment of the testes. When D. simulans PGCs were transplanted into agametic D. melanogaster embryos, the resulting sperm were indistinguishable in length from those of D. simulans donor males and remained significantly shorter than the sperm of D. melanogaster males. Our results suggest that the dramatic diversification in sperm length shaped by post-copulatory sexual selection in the Drosophila lineage has been largely driven by cell-autonomous modifications in the germ line.

Pool-seq (pooled sequencing) combines DNA from multiple individuals prior to sequencing, enabling population-level allele frequency estimation without individual genotyping. When employed in a case-control Genome Wide Association Study (GWAS) framework, pool-seq faces a fundamental power limitation: Errors on allele frequency estimates are inversely proportional to sequence coverage and are large at modest coverage levels. Although this power limitation is appreciated, modestly sized pool-seq GWAS lacking unambiguous hits are often interpreted as showing a polygenic genetic architecture. We illustrate that this inference is unwarranted using empirical data from a Drosophila zinc resistance mapping study. Despite achieving >700× sequencing coverage in case and control pools, a directly ascertained SNP-based GWAS failed to reveal clear evidence for major-effect loci. A unique feature of the dataset is that an advanced intercross multiparent population, with known founders, was employed as the GWAS population. We leverage this unique population structure, in a manner that would not be possible in an outbred panel, to carry out two additional GWASs using imputed haplotype- or SNP-frequency estimates, which in contrast uncover localized regions of major effect. The key difference between approaches lies in statistical power: directly ascertained SNP counts have errors inversely proportional to sequencing coverage, whereas known founder imputation-based approaches can be considerably more accurate. In outbred populations where imputation cannot be used to obtain more accurate allele frequency estimates, substantially higher coverage than currently envisioned may be required to reliably detect modest allele frequency shifts. This work highlights that insufficiently powered GWAS studies can mask simple genetic architectures and create the illusion of polygenicity through statistical noise alone.