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G3: Genes|Genomes|Genetics
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G3: Genes|Genomes|Genetics publishes high-quality, valuable findings, regardless of perceived impact. G3 publishes foundational research that generates useful genetic and genomic information such as genome maps, single gene studies, QTL studies, mutant screens and advances in methods and technology, novel mutant collections, genome-wide association studies (GWAS)  including gene expression, SNP, and CNV studies; exome sequences related to a specific disease but lacking functional follow-up, personal exome and genome sequencing case, disease, and population reports, and more.


Conceived by the Genetics Society of America, with its first issue published June 2011, G3 is fully open access. G3 uses a Creative Commons license that allows the most free use of the data, which anyone can download, analyze, mine, and reuse, provided that the authors of the article receive credit. GSA believes that rapid dissemination of useful data is the necessary foundation for analysis that leads to mechanistic insights. It is our hope is that this strategy will spawn new discovery.


Like GENETICS, G3 is fast—with a 31-day turnaround time from submission to first decision—and rapid time-to-publication. And like GENETICS, G3 manuscripts are thoroughly peer-reviewed, with careful decisions made by practicing scientists. Before publication, G3 articles receive a thorough copy-edit, ensuring that articles enjoy maximum clarity and impact.

Thompson Reuters JCR Impact Factor (2014): 3.198
EigenFactor (2014): 0.00978
Cited Half-life (2014): 2.1 years

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What's Inside the Current Issue of G3



Thursday, January 5 2017 08:46:24 AM

The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata

Bernardo, R. T., Cunha, D. V., Wang, C., Pereira, L., Silva, S., Salazar, S. B., Schroder, M. S., Okamoto, M., Takahashi-Nakaguchi, A., Chibana, H., Aoyama, T., Sa-Correia, I., Azeredo, J., Butler, G., Mira, N. P.

To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata. CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.

Thursday, January 5 2017 08:46:24 AM

Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2

Batovska, J., Cogan, N. O. I., Lynch, S. E., Blacket, M. J.

Internal Transcribed Spacer 2 (ITS2) is a popular DNA barcoding marker; however, in some animal species it is hypervariable and therefore difficult to sequence with traditional methods. With next-generation sequencing (NGS) it is possible to sequence all gene variants despite the presence of single nucleotide polymorphisms (SNPs), insertions/deletions (indels), homopolymeric regions, and microsatellites. Our aim was to compare the performance of Sanger sequencing and NGS amplicon sequencing in characterizing ITS2 in 26 mosquito species represented by 88 samples. The suitability of ITS2 as a DNA barcoding marker for mosquitoes, and its allelic diversity in individuals and species, was also assessed. Compared to Sanger sequencing, NGS was able to characterize the ITS2 region to a greater extent, with resolution within and between individuals and species that was previously not possible. A total of 382 unique sequences (alleles) were generated from the 88 mosquito specimens, demonstrating the diversity present that has been overlooked by traditional sequencing methods. Multiple indels and microsatellites were present in the ITS2 alleles, which were often specific to species or genera, causing variation in sequence length. As a barcoding marker, ITS2 was able to separate all of the species, apart from members of the Culex pipiens complex, providing the same resolution as the commonly used Cytochrome Oxidase I (COI). The ability to cost-effectively sequence hypervariable markers makes NGS an invaluable tool with many applications in the DNA barcoding field, and provides insights into the limitations of previous studies and techniques.

Thursday, January 5 2017 08:46:24 AM

Small RNA Sequencing in Cells and Exosomes Identifies eQTLs and 14q32 as a Region of Active Export

Tsang, E. K., Abell, N. S., Li, X., Anaya, V., Karczewski, K. J., Knowles, D. A., Sierra, R. G., Smith, K. S., Montgomery, S. B.

Exosomes are small extracellular vesicles that carry heterogeneous cargo, including RNA, between cells. Increasing evidence suggests that exosomes are important mediators of intercellular communication and biomarkers of disease. Despite this, the variability of exosomal RNA between individuals has not been well quantified. To assess this variability, we sequenced the small RNA of cells and exosomes from a 17-member family. Across individuals, we show that selective export of miRNAs occurs not only at the level of specific transcripts, but that a cluster of 74 mature miRNAs on chromosome 14q32 is massively exported in exosomes while mostly absent from cells. We also observe more interindividual variability between exosomal samples than between cellular ones and identify four miRNA expression quantitative trait loci shared between cells and exosomes. Our findings indicate that genomically colocated miRNAs can be exported together and highlight the variability in exosomal miRNA levels between individuals as relevant for exosome use as diagnostics.

Thursday, January 5 2017 08:46:24 AM

Bayesian Genomic Prediction with Genotype x Environment Interaction Kernel Models

Cuevas, J., Crossa, J., Montesinos-Lopez, O. A., Burgueno, J., Perez-Rodriguez, P., de los Campos, G.

The phenomenon of genotype x environment (G x E) interaction in plant breeding decreases selection accuracy, thereby negatively affecting genetic gains. Several genomic prediction models incorporating G x E have been recently developed and used in genomic selection of plant breeding programs. Genomic prediction models for assessing multi-environment G x E interaction are extensions of a single-environment model, and have advantages and limitations. In this study, we propose two multi-environment Bayesian genomic models: the first model considers genetic effects $$\left(\mathbf{\boldsymbol{u}}\right)$$ that can be assessed by the Kronecker product of variance–covariance matrices of genetic correlations between environments and genomic kernels through markers under two linear kernel methods, linear (genomic best linear unbiased predictors, GBLUP) and Gaussian (Gaussian kernel, GK). The other model has the same genetic component as the first model $$\left(\mathbf{\boldsymbol{u}}\right)$$ plus an extra component, f, that captures random effects between environments that were not captured by the random effects $$\mathbf{\boldsymbol{u}}.$$ We used five CIMMYT data sets (one maize and four wheat) that were previously used in different studies. Results show that models with G x E always have superior prediction ability than single-environment models, and the higher prediction ability of multi-environment models with $$\mathbf{\boldsymbol{u}}\hspace{0.17em}\hbox{ and }\hspace{0.17em}\mathbf{\boldsymbol{f}}$$ over the multi-environment model with only u occurred 85% of the time with GBLUP and 45% of the time with GK across the five data sets. The latter result indicated that including the random effect f is still beneficial for increasing prediction ability after adjusting by the random effect $$\mathbf{\boldsymbol{u}}.$$

Thursday, January 5 2017 08:46:24 AM

Assortative Mating and Linkage Disequilibrium

Hedrick, P. W.

Assortative mating has been suggested to result in an increase in heritability and additive genetic variance through an increase in linkage disequilibrium. The impact of assortative mating on linkage disequilibrium was explicitly examined for the two-locus model of Wright (1921) and two selective assortative mating models. For the Wright (1921) model, when the proportion of assortative mating was high, positive linkage disequilibrium was generated. However, when the proportion of assortative mating was similar to that found in some studies, the amount of linkage disequilibrium was quite low. In addition, the amount of linkage disequilibrium was independent of the level of recombination. For two selective assortative models, the amount of linkage disequilibrium was a function of the amount of recombination. For these models, the linkage disequilibrium generated was negative mainly because repulsion heterozygotes were favored over coupling heterozygotes. From these findings, the impact of assortative mating on linkage disequilibrium, and consequently heritability and additive genetic variance, appears to be small and model-specific.

Thursday, January 5 2017 08:46:24 AM

The Phenotypic Plasticity of Duplicated Genes in Saccharomyces cerevisiae and the Origin of Adaptations

Mattenberger, F., Sabater-Munoz, B., Toft, C., Fares, M. A.

Gene and genome duplication are the major sources of biological innovations in plants and animals. Functional and transcriptional divergence between the copies after gene duplication has been considered the main driver of innovations . However, here we show that increased phenotypic plasticity after duplication plays a more major role than thought before in the origin of adaptations. We perform an exhaustive analysis of the transcriptional alterations of duplicated genes in the unicellular eukaryote Saccharomyces cerevisiae when challenged with five different environmental stresses. Analysis of the transcriptomes of yeast shows that gene duplication increases the transcriptional response to environmental changes, with duplicated genes exhibiting signatures of adaptive transcriptional patterns in response to stress. The mechanism of duplication matters, with whole-genome duplicates being more transcriptionally altered than small-scale duplicates. The predominant transcriptional pattern follows the classic theory of evolution by gene duplication; with one gene copy remaining unaltered under stress, while its sister copy presents large transcriptional plasticity and a prominent role in adaptation. Moreover, we find additional transcriptional profiles that are suggestive of neo- and subfunctionalization of duplicate gene copies. These patterns are strongly correlated with the functional dependencies and sequence divergence profiles of gene copies. We show that, unlike singletons, duplicates respond more specifically to stress, supporting the role of natural selection in the transcriptional plasticity of duplicates. Our results reveal the underlying transcriptional complexity of duplicated genes and its role in the origin of adaptations.

Thursday, January 5 2017 08:46:24 AM

Cytogenetic and Molecular Characterization of B-Genome Introgression Lines of Brassica napus L.

Dhaliwal, I., Mason, A. S., Banga, S., Bharti, S., Kaur, B., Gurung, A. M., Salisbury, P. A., Batley, J., Banga, S. S.

Brassica napus introgression lines (ILs), having B-genome segments from B. carinata, were assessed genetically for extent of introgression and phenotypically for siliqua shatter resistance. Introgression lines had 7–9% higher DNA content, were meiotically stable, and had almost normal pollen fertility/seed set. Segment introgressions were confirmed by fluorescent genomic in situ hybridization (fl-GISH), SSR analyses, and SNP studies. Genotyping with 48 B-genome specific SSRs detected substitutions from B3, B4, B6, and B7 chromosomes on 39 of the 69 ILs whereas SNP genotyping detected a total of 23 B-segments (≥3 Mb) from B4, B6, and B7 introgressed into 10 of the 19 (C1, C2, C3, C5, C6, C8, C9, A3, A9, A10) chromosomes in 17 ILs. The size of substitutions varied from 3.0 Mb on chromosome A9 (IL59) to 42.44 Mb on chromosome C2 (IL54), ranging from 7 to 83% of the recipient chromosome. Average siliqua strength in ILs was observed to be higher than that of B. napus parents (2.2–6.0 vs. 1.9–4.0 mJ) while siliqua strength in some of the lines was almost equal to that of the donor parent B. carinata (6.0 vs.7.2 mJ). These ILs, with large chunks of substituted B-genome, can prove to be a useful prebreeding resource for germplasm enhancement in B. napus, especially for siliqua shatter resistance.

Thursday, January 5 2017 08:46:24 AM

Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila Using a Co-CRISPR Strategy

Kane, N. S., Vora, M., Varre, K. J., Padgett, R. W.

Genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease (Cas9) enables specific genetic modifications, including deletions, insertions, and substitutions in numerous organisms, such as the fruit fly Drosophila melanogaster. One challenge of the CRISPR/Cas9 system can be the laborious and time-consuming screening required to find CRISPR-induced modifications due to a lack of an obvious phenotype and low frequency after editing. Here we apply the successful co-CRISPR technique in Drosophila to simultaneously target a gene of interest and a marker gene, ebony, which is a recessive gene that produces dark body color and has the further advantage of not being a commonly used transgenic marker. We found that Drosophila broods containing higher numbers of CRISPR-induced ebony mutations ("jackpot" lines) are significantly enriched for indel events in a separate gene of interest, while broods with few or no ebony offspring showed few mutations in the gene of interest. Using two different PAM sites in our gene of interest, we report that ~61% (52–70%) of flies from the ebony-enriched broods had an indel in DNA near either PAM site. Furthermore, this marker mutation system may be useful in detecting the less frequent homology-directed repair events, all of which occurred in the ebony-enriched broods. By focusing on the broods with a significant number of ebony flies, successful identification of CRISPR-induced events is much faster and more efficient. The co-CRISPR technique we present significantly improves the screening efficiency in identification of genome-editing events in Drosophila.

Thursday, January 5 2017 08:46:24 AM

Candida albicans Is Resistant to Polyglutamine Aggregation and Toxicity

Leach, M. D., Kim, T., DiGregorio, S. E., Collins, C., Zhang, Z., Duennwald, M. L., Cowen, L. E.

Disruption of protein quality control can be detrimental, having toxic effects on single cell organisms and contributing to neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s in humans. Here, we examined the effects of polyglutamine (polyQ) aggregation in a major fungal pathogen of humans, Candida albicans, with the goal of identifying new approaches to disable this fungus. However, we discovered that expression of polyQ stretches up to 230Q had no effect on C. albicans ability to grow and withstand proteotoxic stress. Bioinformatics analysis demonstrates that C. albicans has a similarly glutamine-rich proteome to the unicellular fungus Saccharomyces cerevisiae, which exhibits polyQ toxicity with as few as 72Q. Surprisingly, global transcriptional profiles indicated no significant change upon induction of up to 230Q. Proteomic analysis highlighted two key interactors of 230Q, Sis1 and Sgt2; however, loss of either protein had no additional effect on C. albicans toxicity. Our data suggest that C. albicans has evolved powerful mechanisms to overcome the toxicity associated with aggregation-prone proteins, providing a unique model for studying polyQ-associated diseases.

Thursday, January 5 2017 08:46:24 AM

A New Chicken Genome Assembly Provides Insight into Avian Genome Structure

Warren, W. C., Hillier, L. W., Tomlinson, C., Minx, P., Kremitzki, M., Graves, T., Markovic, C., Bouk, N., Pruitt, K. D., Thibaud-Nissen, F., Schneider, V., Mansour, T. A., Brown, C. T., Zimin, A., Hawken, R., Abrahamsen, M., Pyrkosz, A. B., Morisson, M., Fillon, V., Vignal, A., Chow, W., Howe, K., Fulton, J. E., Miller, M. M., Lovell, P., Mello, C. V., Wirthlin, M., Mason, A. S., Kuo, R., Burt, D. W., Dodgson, J. B., Cheng, H. H.

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts.

Thursday, January 5 2017 08:46:24 AM

Imputation-Based Fine-Mapping Suggests That Most QTL in an Outbred Chicken Advanced Intercross Body Weight Line Are Due to Multiple, Linked Loci

Brandt, M., Ahsan, M., Honaker, C. F., Siegel, P. B., Carlborg, O.

The Virginia chicken lines have been divergently selected for juvenile body weight for more than 50 generations. Today, the high- and low-weight lines show a >12-fold difference for the selected trait, 56-d body weight. These lines provide unique opportunities to study the genetic architecture of long-term, single-trait selection. Previously, several quantitative trait loci (QTL) contributing to weight differences between the lines were mapped in an F2-cross between them, and these were later replicated and fine-mapped in a nine-generation advanced intercross of them. Here, we explore the possibility to further increase the fine-mapping resolution of these QTL via a pedigree-based imputation strategy that aims to better capture the genetic diversity in the divergently selected, but outbred, founder lines. The founders of the intercross were high-density genotyped, and then pedigree-based imputation was used to assign genotypes throughout the pedigree. Imputation increased the marker density 20-fold in the selected QTL, providing 6911 markers for the subsequent analysis. Both single-marker association and multi-marker backward-elimination analyses were used to explore regions associated with 56-d body weight. The approach revealed several statistically and population structure independent associations and increased the mapping resolution. Further, most QTL were also found to contain multiple independent associations to markers that were not fixed in the founder populations, implying a complex underlying architecture due to the combined effects of multiple, linked loci perhaps located on independent haplotypes that still segregate in the selected lines.

Thursday, January 5 2017 08:46:24 AM

The Neurospora Transcription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion

Dekhang, R., Wu, C., Smith, K. M., Lamb, T. M., Peterson, M., Bredeweg, E. L., Ibarra, O., Emerson, J. M., Karunarathna, N., Lyubetskaya, A., Azizi, E., Hurley, J. M., Dunlap, J. C., Galagan, J. E., Freitag, M., Sachs, M. S., Bell-Pedersen, D.

Light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. Toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor ADV-1 in Neurospora crassa. A large proportion of ADV-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in development, metabolism, cell growth, and cell fusion. We show that ADV-1 is necessary for transducing light and/or temporal information to its immediate downstream targets, including controlling rhythms in genes critical to somatic cell fusion. However, while ADV-1 targets are altered in predictable ways in adv-1 cells in response to light, this is not always the case for rhythmic target gene expression. These data suggest that a complex regulatory network downstream of ADV-1 functions to generate distinct temporal dynamics of target gene expression relative to the central clock mechanism.

Thursday, January 5 2017 08:46:24 AM

iSeq: A New Double-Barcode Method for Detecting Dynamic Genetic Interactions in Yeast

Jaffe, M., Sherlock, G., Levy, S. F.

Systematic screens for genetic interactions are a cornerstone of both network and systems biology. However, most screens have been limited to characterizing interaction networks in a single environment. Moving beyond this static view of the cell requires a major technological advance to increase the throughput and ease of replication in these assays. Here, we introduce iSeq—a platform to build large double barcode libraries and rapidly assay genetic interactions across environments. We use iSeq in yeast to measure fitness in three conditions of nearly 400 clonal strains, representing 45 possible single or double gene deletions, including multiple replicate strains per genotype. We show that iSeq fitness and interaction scores are highly reproducible for the same clonal strain across replicate cultures. However, consistent with previous work, we find that replicates with the same putative genotype have highly variable genetic interaction scores. By whole-genome sequencing 102 of our strains, we find that segregating variation and de novo mutations, including aneuploidy, occur frequently during strain construction, and can have large effects on genetic interaction scores. Additionally, we uncover several new environment-dependent genetic interactions, suggesting that barcode-based genetic interaction assays have the potential to significantly expand our knowledge of genetic interaction networks.

Thursday, January 5 2017 08:46:24 AM

The Physical Genome Mapping of Anopheles albimanus Corrected Scaffold Misassemblies and Identified Interarm Rearrangements in Genus Anopheles

Artemov, G. N., Peery, A. N., Jiang, X., Tu, Z., Stegniy, V. N., Sharakhova, M. V., Sharakhov, I. V.

The genome of the Neotropical malaria vector Anopheles albimanus was sequenced as part of the 16 Anopheles Genomes Project published in 2015. The draft assembly of this species consisted of 204 scaffolds with an N50 scaffold size of 18.1 Mb and a total assembly size of 170.5 Mb. It was among the smallest genomes with the longest scaffolds in the 16 Anopheles species cluster, making An. albimanus the logical choice for anchoring the genome assembly to chromosomes. In this study, we developed a high-resolution cytogenetic photomap with completely straightened polytene chromosomes from the salivary glands of the mosquito larvae. Based on this photomap, we constructed a chromosome-based genome assembly using fluorescent in situ hybridization of PCR-amplified DNA probes. Our physical mapping, assisted by an ortholog-based bioinformatics approach, identified and corrected nine misassemblies in five large genomic scaffolds. Misassemblies mostly occurred in junctions between contigs. Our comparative analysis of scaffolds with the An. gambiae genome detected multiple genetic exchanges between pericentromeric regions of chromosomal arms caused by partial-arm translocations. The final map consists of 40 ordered genomic scaffolds and corrected fragments of misassembled scaffolds. The An. albimanus physical map comprises 98.2% of the total genome assembly and represents the most complete genome map among mosquito species. This study demonstrates that physical mapping is a powerful tool for correcting errors in draft genome assemblies and for creating chromosome-anchored reference genomes.

Thursday, January 5 2017 08:46:24 AM

Regulatory Architecture of Gene Expression Variation in the Threespine Stickleback Gasterosteus aculeatus

Pritchard, V. L., Viitaniemi, H. M., McCairns, R. J. S., Merila, J., Nikinmaa, M., Primmer, C. R., Leder, E. H.

Much adaptive evolutionary change is underlain by mutational variation in regions of the genome that regulate gene expression rather than in the coding regions of the genes themselves. An understanding of the role of gene expression variation in facilitating local adaptation will be aided by an understanding of underlying regulatory networks. Here, we characterize the genetic architecture of gene expression variation in the threespine stickleback (Gasterosteus aculeatus), an important model in the study of adaptive evolution. We collected transcriptomic and genomic data from 60 half-sib families using an expression microarray and genotyping-by-sequencing, and located expression quantitative trait loci (eQTL) underlying the variation in gene expression in liver tissue using an interval mapping approach. We identified eQTL for several thousand expression traits. Expression was influenced by polymorphism in both cis- and trans-regulatory regions. Trans-eQTL clustered into hotspots. We did not identify master transcriptional regulators in hotspot locations: rather, the presence of hotspots may be driven by complex interactions between multiple transcription factors. One observed hotspot colocated with a QTL recently found to underlie salinity tolerance in the threespine stickleback. However, most other observed hotspots did not colocate with regions of the genome known to be involved in adaptive divergence between marine and freshwater habitats.

Thursday, January 5 2017 08:46:24 AM

Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

Yamamoto, T., Fujimura-Kamada, K., Shioji, E., Suzuki, R., Tanaka, K.

Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p. They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the cdc50 mutant. We identified a mutation of YMR010W encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene CFS1 (cdc fifty suppressor 1). GFP-tagged Cfs1p was partially colocalized with Drs2p and Neo1p to endosomal/late Golgi membranes. Interestingly, the cfs1 mutation suppressed growth defects in all flippase mutants. Accordingly, defects in membrane trafficking in the flippase mutants were also suppressed. These results suggest that Cfs1p and flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the cfs1 mutation changed PE asymmetry in the plasma membrane. Cfs1p may thus be a novel regulator of phospholipid asymmetry.

Thursday, January 5 2017 08:46:24 AM

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

Shen, H., Strunks, G. D., Klemann, B. J. P. M., Hooykaas, P. J. J., de Pater, S.

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase -mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.

Thursday, January 5 2017 08:46:24 AM

Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation

Zhao, D., Hamilton, J. P., Hardigan, M., Yin, D., He, T., Vaillancourt, B., Reynoso, M., Pauluzzi, G., Funkhouser, S., Cui, Y., Bailey-Serres, J., Jiang, J., Buell, C. R., Jiang, N.

Gene expression is controlled at transcriptional and post-transcriptional levels including decoding of messenger RNA (mRNA) into polypeptides via ribosome-mediated translation. Translational regulation has been intensively studied in the model dicot plant Arabidopsis thaliana, and in this study, we assessed the translational status [proportion of steady-state mRNA associated with ribosomes] of mRNAs by Translating Ribosome Affinity Purification followed by mRNA-sequencing (TRAP-seq) in rice (Oryza sativa), a model monocot plant and the most important food crop. A survey of three tissues found that most transcribed rice genes are translated whereas few transposable elements are associated with ribosomes. Genes with short and GC-rich coding regions are overrepresented in ribosome-associated mRNAs, suggesting that the GC-richness characteristic of coding sequences in grasses may be an adaptation that favors efficient translation. Transcripts with retained introns and extended 5' untranslated regions are underrepresented on ribosomes, and rice genes belonging to different evolutionary lineages exhibited differential enrichment on the ribosomes that was associated with GC content. Genes involved in photosynthesis and stress responses are preferentially associated with ribosomes, whereas genes in epigenetic regulation pathways are the least enriched on ribosomes. Such variation is more dramatic in rice than that in Arabidopsis and is correlated with the wide variation of GC content of transcripts in rice. Taken together, variation in the translation status of individual transcripts reflects important mechanisms of gene regulation, which may have a role in evolution and diversification.

Thursday, January 5 2017 08:46:24 AM

Time-Course Analysis of Gene Expression During the Saccharomyces cerevisiae Hypoxic Response

Bendjilali, N., MacLeon, S., Kalra, G., Willis, S. D., Hossian, A. K. M. N., Avery, E., Wojtowicz, O., Hickman, M. J.

Many cells experience hypoxia, or low oxygen, and respond by dramatically altering gene expression. In the yeast Saccharomyces cerevisiae, genes that respond are required for many oxygen-dependent cellular processes, such as respiration, biosynthesis, and redox regulation. To more fully characterize the global response to hypoxia, we exposed yeast to hypoxic conditions, extracted RNA at different times, and performed RNA sequencing (RNA-seq) analysis. Time-course statistical analysis revealed hundreds of genes that changed expression by up to 550-fold. The genes responded with varying kinetics suggesting that multiple regulatory pathways are involved. We identified most known oxygen-regulated genes and also uncovered new regulated genes. Reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed that the lysine methyltransferase EFM6 and the recombinase DMC1, both conserved in humans, are indeed oxygen-responsive. Looking more broadly, oxygen-regulated genes participate in expected processes like respiration and lipid metabolism, but also in unexpected processes like amino acid and vitamin metabolism. Using principle component analysis, we discovered that the hypoxic response largely occurs during the first 2 hr and then a new steady-state expression state is achieved. Moreover, we show that the oxygen-dependent genes are not part of the previously described environmental stress response (ESR) consisting of genes that respond to diverse types of stress. While hypoxia appears to cause a transient stress, the hypoxic response is mostly characterized by a transition to a new state of gene expression. In summary, our results reveal that hypoxia causes widespread and complex changes in gene expression to prepare the cell to function with little or no oxygen.

Thursday, January 5 2017 08:46:24 AM

Dissecting Gene Expression Changes Accompanying a Ploidy-Based Phenotypic Switch

Cromie, G. A., Tan, Z., Hays, M., Jeffery, E. W., Dudley, A. M.

Aneuploidy, a state in which the chromosome number deviates from a multiple of the haploid count, significantly impacts human health. The phenotypic consequences of aneuploidy are believed to arise from gene expression changes associated with the altered copy number of genes on the aneuploid chromosomes. To dissect the mechanisms underlying altered gene expression in aneuploids, we used RNA-seq to measure transcript abundance in colonies of the haploid Saccharomyces cerevisiae strain F45 and two aneuploid derivatives harboring disomies of chromosomes XV and XVI. F45 colonies display complex "fluffy" morphologies, while the disomic colonies are smooth, resembling laboratory strains. Our two disomes displayed similar transcriptional profiles, a phenomenon not driven by their shared smooth colony morphology nor simply by their karyotype. Surprisingly, the environmental stress response (ESR) was induced in F45, relative to the two disomes. We also identified genes whose expression reflected a nonlinear interaction between the copy number of a transcriptional regulatory gene on chromosome XVI, DIG1, and the copy number of other chromosome XVI genes. DIG1 and the remaining chromosome XVI genes also demonstrated distinct contributions to the effect of the chromosome XVI disome on ESR gene expression. Expression changes in aneuploids appear to reflect a mixture of effects shared between different aneuploidies and effects unique to perturbing the copy number of particular chromosomes, including nonlinear copy number interactions between genes. The balance between these two phenomena is likely to be genotype- and environment-specific.

Thursday, January 5 2017 08:46:24 AM

Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis

Ho, N. R. Y., Usmani, A. R., Yin, Y., Ma, L., Conrad, D. F.

Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6–9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA) constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi) against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that can act as potent and highly specific male contraceptives.

Thursday, January 5 2017 08:46:24 AM

Caenorhabditis elegans DAF-2 as a Model for Human Insulin Receptoropathies

Bulger, D. A., Fukushige, T., Yun, S., Semple, R. K., Hanover, J. A., Krause, M. W.

Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP) and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2. CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways.

Thursday, January 5 2017 08:46:25 AM

First in Vivo Batrachochytrium dendrobatidis Transcriptomes Reveal Mechanisms of Host Exploitation, Host-Specific Gene Expression, and Expressed Genotype Shifts

Ellison, A. R., DiRenzo, G. V., McDonald, C. A., Lips, K. R., Zamudio, K. R.

For generalist pathogens, host species represent distinct selective environments, providing unique challenges for resource acquisition and defense from host immunity, potentially resulting in host-dependent differences in pathogen fitness. Gene expression modulation should be advantageous, responding optimally to a given host and mitigating the costs of generalism. Batrachochytrium dendrobatidis (Bd), a fungal pathogen of amphibians, shows variability in pathogenicity among isolates, and within-strain virulence changes rapidly during serial passages through artificial culture. For the first time, we characterize the transcriptomic profile of Bd in vivo, using laser-capture microdissection. Comparison of Bd transcriptomes (strain JEL423) in culture and in two hosts (Atelopus zeteki and Hylomantis lemur), reveals >2000 differentially expressed genes that likely include key Bd defense and host exploitation mechanisms. Variation in Bd transcriptomes from different amphibian hosts demonstrates shifts in pathogen resource allocation. Furthermore, expressed genotype variant frequencies of Bd populations differ between culture and amphibian skin, and among host species, revealing potential mechanisms underlying rapid changes in virulence and the possibility that amphibian community composition shapes Bd evolutionary trajectories. Our results provide new insights into how changes in gene expression and infecting population genotypes can be key to the success of a generalist fungal pathogen.

Thursday, January 5 2017 08:46:25 AM

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

Herricks, T., Dilworth, D. J., Mast, F. D., Li, S., Smith, J. J., Ratushny, A. V., Aitchison, J. D.

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAY extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.

Thursday, January 5 2017 08:46:25 AM

Correlations of Genotype with Climate Parameters Suggest Caenorhabditis elegans Niche Adaptations

Evans, K. S., Zhao, Y., Brady, S. C., Long, L., McGrath, P. T., Andersen, E. C.

Species inhabit a variety of environmental niches, and the adaptation to a particular niche is often controlled by genetic factors, including gene-by-environment interactions. The genes that vary in order to regulate the ability to colonize a niche are often difficult to identify, especially in the context of complex ecological systems and in experimentally uncontrolled natural environments. Quantitative genetic approaches provide an opportunity to investigate correlations between genetic factors and environmental parameters that might define a niche. Previously, we have shown how a collection of 208 whole-genome sequenced wild Caenorhabditis elegans can facilitate association mapping approaches. To correlate climate parameters with the variation found in this collection of wild strains, we used geographic data to exhaustively curate daily weather measurements in short-term (3 month), middle-term (one year), and long-term (three year) durations surrounding the date of strain isolation. These climate parameters were used as quantitative traits in association mapping approaches, where we identified 11 quantitative trait loci (QTL) for three climatic variables: elevation, relative humidity, and average temperature. We then narrowed the genomic interval of interest to identify gene candidates with variants potentially underlying phenotypic differences. Additionally, we performed two-strain competition assays at high and low temperatures to validate a QTL that could underlie adaptation to temperature and found suggestive evidence supporting that hypothesis.

Thursday, January 5 2017 08:46:25 AM

Genotyping-by-Sequencing-Based Investigation of the Genetic Architecture Responsible for a ~Sevenfold Increase in Soybean Seed Stearic Acid

Heim, C. B., Gillman, J. D.

Soybean oil is highly unsaturated but oxidatively unstable, rendering it nonideal for food applications. Until recently, the majority of soybean oil underwent partial chemical hydrogenation, which produces trans fats as an unavoidable consequence. Dietary intake of trans fats and most saturated fats are conclusively linked to negative impacts on cholesterol levels and cardiovascular health. Two major soybean oil breeding targets are: (1) to reduce or eliminate the need for chemical hydrogenation, and (2) to replace the functional properties of partially hydrogenated soybean oil. One potential solution is the elevation of seed stearic acid, a saturated fat which has no negative impacts on cardiovascular health, from 3 to 4% in typical cultivars to > 20% of the seed oil. We performed QTL analysis of a population developed by crossing two mutant lines, one with a missense mutation affecting a stearoyl-acyl-carrier protein desaturase gene resulting in ~11% seed stearic acid crossed to another mutant, A6, which has 24–28% seed stearic acid. Genotyping-by-sequencing (GBS)-based QTL mapping identified 21 minor and major effect QTL for six seed oil related traits and plant height. The inheritance of a large genomic deletion affecting chromosome 14 is the basis for largest effect QTL, resulting in ~18% seed stearic acid. This deletion contains SACPD-C and another gene(s); loss of both genes boosts seed stearic acid levels to ≥ 18%. Unfortunately, this genomic deletion has been shown in previous studies to be inextricably correlated with reduced seed yield. Our results will help inform and guide ongoing breeding efforts to improve soybean oil oxidative stability.

Thursday, January 5 2017 08:46:25 AM

Identification of Genes Mediating Drosophila Follicle Cell Progenitor Differentiation by Screening for Modifiers of GAL4::UAS Variegation

Lee, M.-C., Skora, A. D., Spradling, A. C.

The Drosophila melanogaster ovarian follicle cell lineage provides a powerful system for investigating how epigenetic changes contribute to differentiation. Downstream from an epithelial stem cell, follicle progenitors undergo nine mitotic cell cycles before transitioning to the endocycle and initiating differentiation. During their proliferative phase, follicle progenitors experience Lsd1-dependent changes in epigenetic stability that can be monitored using GAL4::UAS variegation. Eventually, follicle progenitors acquire competence to respond to Delta, a Notch ligand present in the environment, which signals them to cease division and initiate differentiation. The time required to acquire competence determines the duration of mitotic cycling and hence the final number of follicle cells. We carried out a screen for dominant modifiers of variegation spanning nearly 70% of Drosophila euchromatin to identify new genes influencing follicle progenitor epigenetic maturation. The eight genes found include chromatin modifiers, but also cell cycle regulators and transcription factors. Five of the modifier genes accelerate the acquisition of progenitor competence and reduce follicle cell number, however, the other three genes affect follicle cell number in an unexpected manner.

Thursday, January 5 2017 08:46:25 AM