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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, September 8 2016 08:30:55 AM

Retrotransposon Proliferation Coincident with the Evolution of Dioecy in Asparagus

Harkess, A., Mercati, F., Abbate, L., McKain, M., Pires, J. C., Sala, T., Sunseri, F., Falavigna, A., Leebens-Mack, J.


Current phylogenetic sampling reveals that dioecy and an XY sex chromosome pair evolved once, or possibly twice, in the genus Asparagus. Although there appear to be some lineage-specific polyploidization events, the base chromosome number of 2n = 2= 20 is relatively conserved across the Asparagus genus. Regardless, dioecious species tend to have larger genomes than hermaphroditic species. Here, we test whether this genome size expansion in dioecious species is related to a polyploidization and subsequent chromosome fusion, or to retrotransposon proliferation in dioecious species. We first estimate genome sizes, or use published values, for four hermaphrodites and four dioecious species distributed across the phylogeny, and show that dioecious species typically have larger genomes than hermaphroditic species. Utilizing a phylogenomic approach, we find no evidence for ancient polyploidization contributing to increased genome sizes of sampled dioecious species. We do find support for an ancient whole genome duplication (WGD) event predating the diversification of the Asparagus genus. Repetitive DNA content of the four hermaphroditic and four dioecious species was characterized based on randomly sampled whole genome shotgun sequencing, and common elements were annotated. Across our broad phylogenetic sampling, Ty-1 Copia retroelements, in particular, have undergone a marked proliferation in dioecious species. In the absence of a detectable WGD event, retrotransposon proliferation is the most likely explanation for the precipitous increase in genome size in dioecious Asparagus species.



Thursday, September 8 2016 08:30:55 AM

An Inversion Disrupting FAM134B Is Associated with Sensory Neuropathy in the Border Collie Dog Breed

Forman, O. P., Hitti, R. J., Pettitt, L., Jenkins, C. A., OBrien, D. P., Shelton, G. D., De Risio, L., Quintana, R. G., Beltran, E., Mellersh, C.


Sensory neuropathy in the Border Collie is a severe neurological disorder caused by the degeneration of sensory and, to a lesser extent, motor nerve cells with clinical signs starting between 2 and 7 months of age. Using a genome-wide association study approach with three cases and 170 breed matched controls, a suggestive locus for sensory neuropathy was identified that was followed up using a genome sequencing approach. An inversion disrupting the candidate gene FAM134B was identified. Genotyping of additional cases and controls and RNAseq analysis provided strong evidence that the inversion is causal. Evidence of cryptic splicing resulting in novel exon transcription for FAM134B was identified by RNAseq experiments. This investigation demonstrates the identification of a novel sensory neuropathy associated mutation, by mapping using a minimal set of cases and subsequent genome sequencing. Through mutation screening, it should be possible to reduce the frequency of or completely eliminate this debilitating condition from the Border Collie breed population.



Thursday, September 8 2016 08:30:55 AM

Genomic Signatures of North American Soybean Improvement Inform Diversity Enrichment Strategies and Clarify the Impact of Hybridization

Vaughn, J. N., Li, Z.


Crop improvement represents a long-running experiment in artificial selection on a complex trait, namely yield. How such selection relates to natural populations is unclear, but the analysis of domesticated populations could offer insights into the relative role of selection, drift, and recombination in all species facing major shifts in selective regimes. Because of the extreme autogamy exhibited by soybean (Glycine max), many "immortalized" genotypes of elite varieties spanning the last century have been preserved and characterized using ~50,000 single nucleotide polymorphic (SNP) markers. Also due to autogamy, the history of North American soybean breeding can be roughly divided into pre- and posthybridization eras, allowing for direct interrogation of the role of recombination in improvement and selection. Here, we report on genome-wide characterization of the structure and history of North American soybean populations and the signature of selection in these populations. Supporting previous work, we find that maturity defines population structure. Though the diversity of North American ancestors is comparable to available landraces, prehybridization line selections resulted in a clonal structure that dominated early breeding and explains many of the reductions in diversity found in the initial generations of soybean hybridization. The rate of allele frequency change does not deviate sharply from neutral expectation, yet some regions bare hallmarks of strong selection, suggesting a highly variable range of selection strengths biased toward weak effects. We also discuss the importance of haplotypes as units of analysis when complex traits fall under novel selection regimes.



Thursday, September 8 2016 08:30:55 AM

Microsporidia Intracellular Development Relies on Myc Interaction Network Transcription Factors in the Host

Botts, M. R., Cohen, L. B., Probert, C. S., Wu, F., Troemel, E. R.


Microsporidia are ubiquitous parasites that infect a wide range of animal hosts, and these fungal-related microbes undergo their entire replicative lifecycle inside of host cells. Despite being widespread in the environment and causing medical and agricultural harm, virtually nothing is known about the host factors important to facilitate their growth and development inside of host cells. Here, we perform a genetic screen to identify host transcription factors important for development of the microsporidian pathogen Nematocida parisii inside intestinal cells of its natural host, the nematode Caenorhabditis elegans. Through this screen, we identified the C. elegans Myc family of transcription factors as key host regulators of microsporidia growth and development. The Mad-like transcription factor MDL-1, and the Max-like transcription factors MXL-1 and MXL-2 promote pathogen levels, while the Myc-Mondo-like transcription factor MML-1 inhibits pathogen levels. We used epistasis analysis to show that MDL-1 and MXL-1, which are thought to function as a heterodimer, appear to be acting canonically. In contrast, MXL-2 and MML-1, which are also thought to function as a heterodimer, appear to be acting in separate pathways (noncanonically) in the context of pathogen infection. We also found that both MDL-1::GFP and MML-1::GFP are expressed in intestinal cells during infection. These findings provide novel insight into the host transcription factors that regulate microsporidia development.



Thursday, September 8 2016 08:30:55 AM

Genetic Analysis and QTL Detection on Fiber Traits Using Two Recombinant Inbred Lines and Their Backcross Populations in Upland Cotton

Shang, L., Wang, Y., Wang, X., Liu, F., Abduweli, A., Cai, S., Li, Y., Ma, L., Wang, K., Hua, J.


Cotton fiber, a raw natural fiber material, is widely used in the textile industry. Understanding the genetic mechanism of fiber traits is helpful for fiber quality improvement. In the present study, the genetic basis of fiber quality traits was explored using two recombinant inbred lines (RILs) and corresponding backcross (BC) populations under multiple environments in Upland cotton based on marker analysis. In backcross populations, no significant correlation was observed between marker heterozygosity and fiber quality performance and it suggested that heterozygosity was not always necessarily advantageous for the high fiber quality. In two hybrids, 111 quantitative trait loci (QTL) for fiber quality were detected using composite interval mapping, in which 62 new stable QTL were simultaneously identified in more than one environment or population. QTL detected at the single-locus level mainly showed additive effect. In addition, a total of 286 digenic interactions (E-QTL) and their environmental interactions [QTL x environment interactions (QEs)] were detected for fiber quality traits by inclusive composite interval mapping. QE effects should be considered in molecular marker-assisted selection breeding. On average, the E-QTL explained a larger proportion of the phenotypic variation than the main-effect QTL did. It is concluded that the additive effect of single-locus and epistasis with few detectable main effects play an important role in controlling fiber quality traits in Upland cotton.



Thursday, September 8 2016 08:30:55 AM

A Genomic Bayesian Multi-trait and Multi-environment Model

Montesinos-Lopez, O. A., Montesinos-Lopez, A., Crossa, J., Toledo, F. H., Perez-Hernandez, O., Eskridge, K. M., Rutkoski, J.


When information on multiple genotypes evaluated in multiple environments is recorded, a multi-environment single trait model for assessing genotype x environment interaction (G x E) is usually employed. Comprehensive models that simultaneously take into account the correlated traits and trait x genotype x environment interaction (T x G x E) are lacking. In this research, we propose a Bayesian model for analyzing multiple traits and multiple environments for whole-genome prediction (WGP) model. For this model, we used Half-$$\mathit{t}$$ priors on each standard deviation term and uniform priors on each correlation of the covariance matrix. These priors were not informative and led to posterior inferences that were insensitive to the choice of hyper-parameters. We also developed a computationally efficient Markov Chain Monte Carlo (MCMC) under the above priors, which allowed us to obtain all required full conditional distributions of the parameters leading to an exact Gibbs sampling for the posterior distribution. We used two real data sets to implement and evaluate the proposed Bayesian method and found that when the correlation between traits was high (>0.5), the proposed model (with unstructured variance–covariance) improved prediction accuracy compared to the model with diagonal and standard variance–covariance structures. The R-software package Bayesian Multi-Trait and Multi-Environment (BMTME) offers optimized C++ routines to efficiently perform the analyses.



Thursday, September 8 2016 08:30:55 AM

Identification of QTLs Associated with Virulence Related Traits and Drug Resistance in Cryptococcus neoformans

Vogan, A. A., Khankhet, J., Samarasinghe, H., Xu, J.


Cryptococcus neoformans is a basidiomycete fungus capable of causing deadly meningoenchephilitis, primarily in immunocompromised individuals. Formerly, C. neoformans was composed of two divergent lineages, but these have recently been elevated to species status, now C. neoformans (formerly C. neoformans var. grubii) and C. deneoformans (formerly C. neoformans var. neoformans). While both species can cause deadly infections in humans, C. neoformans is much more prevalent in clinical settings than C. deneoformans. However, the genetic factors contributing to their significant differences in virulence remain largely unknown. Quantitative trait locus (QTL) mapping is a powerful tool that can be used to identify genomic regions associated with phenotypic differences between strains. Here, we analyzed a hybrid cross between these two species and identified a total of 23 QTL, including five for melanin production, six for cell size, one for cell wall thickness, five for the frequency of capsule production, three for minimal inhibitory concentration (MIC) of fluconazole in broth, and three for MIC on solid medium. For the fluconazole resistance-associated QTL, three showed environment and/or concentration-specific effects. Our results provide a large number of candidate gene regions from which to explore the molecular bases for phenotypic differences between C. neoformans and C. deneoformans.



Thursday, September 8 2016 08:30:55 AM

Covariance Between Genotypic Effects and its Use for Genomic Inference in Half-Sib Families

Wittenburg, D., Teuscher, F., Klosa, J., Reinsch, N.


In livestock, current statistical approaches utilize extensive molecular data, e.g., single nucleotide polymorphisms (SNPs), to improve the genetic evaluation of individuals. The number of model parameters increases with the number of SNPs, so the multicollinearity between covariates can affect the results obtained using whole genome regression methods. In this study, dependencies between SNPs due to linkage and linkage disequilibrium among the chromosome segments were explicitly considered in methods used to estimate the effects of SNPs. The population structure affects the extent of such dependencies, so the covariance among SNP genotypes was derived for half-sib families, which are typical in livestock populations. Conditional on the SNP haplotypes of the common parent (sire), the theoretical covariance was determined using the haplotype frequencies of the population from which the individual parent (dam) was derived. The resulting covariance matrix was included in a statistical model for a trait of interest, and this covariance matrix was then used to specify prior assumptions for SNP effects in a Bayesian framework. The approach was applied to one family in simulated scenarios (few and many quantitative trait loci) and using semireal data obtained from dairy cattle to identify genome segments that affect performance traits, as well as to investigate the impact on predictive ability. Compared with a method that does not explicitly consider any of the relationship among predictor variables, the accuracy of genetic value prediction was improved by 10–22%. The results show that the inclusion of dependence is particularly important for genomic inference based on small sample sizes.



Thursday, September 8 2016 08:30:55 AM

A Comparative Analysis of 5-Azacytidine- and Zebularine-Induced DNA Demethylation

Griffin, P. T., Niederhuth, C. E., Schmitz, R. J.


The nonmethylable cytosine analogs, 5-azacytidine and zebularine, are widely used to inhibit DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that both inhibitor treatments result in nearly indistinguishable patterns of genome-wide DNA methylation and that 5-azacytidine had a slightly greater demethylating effect at higher concentrations across the genome. Transcriptome analyses revealed a substantial number of upregulated genes, with an overrepresentation of transposable element genes, in particular CACTA-like elements. This demonstrates that chemical demethylating agents have a disproportionately large effect on loci that are otherwise silenced by DNA methylation.



Thursday, September 8 2016 08:30:55 AM

Sources of Error in Mammalian Genetic Screens

Sack, L. M., Davoli, T., Xu, Q., Li, M. Z., Elledge, S. J.


Genetic screens are invaluable tools for dissection of biological phenomena. Optimization of such screens to enhance discovery of candidate genes and minimize false positives is thus a critical aim. Here, we report several sources of error common to pooled genetic screening techniques used in mammalian cell culture systems, and demonstrate methods to eliminate these errors. We find that reverse transcriptase-mediated recombination during retroviral replication can lead to uncoupling of molecular tags, such as DNA barcodes (BCs), from their associated library elements, leading to chimeric proviral genomes in which BCs are paired to incorrect ORFs, shRNAs, etc. This effect depends on the length of homologous sequence between unique elements, and can be minimized with careful vector design. Furthermore, we report that residual plasmid DNA from viral packaging procedures can contaminate transduced cells. These plasmids serve as additional copies of the PCR template during library amplification, resulting in substantial inaccuracies in measurement of initial reference populations for screen normalization. The overabundance of template in some samples causes an imbalance between PCR cycles of contaminated and uncontaminated samples, which results in a systematic artifactual depletion of GC-rich library elements. Elimination of contaminating plasmid DNA using the bacterial endonuclease Benzonase can restore faithful measurements of template abundance and minimize GC bias.



Thursday, September 8 2016 08:30:55 AM

Whole Genome Comparison of Thermus sp. NMX2.A1 Reveals Principal Carbon Metabolism Differences with Closest Relation Thermus scotoductus SA-01

Muller, W. J., Tlalajoe, N., Cason, E. D., Litthauer, D., Reva, O., Brzuszkiewicz, E., van Heerden, E.


Genome sequencing of the yellow-pigmented, thermophilic bacterium Thermus sp. NMX2.A1 resulted in a 2.29 Mb draft genome that encodes for 2312 proteins. The genetic relationship between various strains from the genus Thermus was assessed based on phylogenomic analyses using a concatenated set of conserved proteins. The resulting phylogenetic tree illustrated that Thermus sp. NMX2 A.1 clusters together with Thermus scotoductus SA-01, despite being isolated from vastly different geographical locations. The close evolutionary relationship and metabolic parallels between the two strains has previously been recognized; however, neither strain’s genome data were available at that point in time. Genomic comparison of the Thermus sp. NMX2.A1 and T. scotoductus SA-01, as well as other closely related Thermus strains, revealed a high degree of synteny at both the genomic and proteomic level, with processes such as denitrification and natural cell competence appearing to be conserved. However, despite this high level of similarity, analysis revealed a complete, putative Calvin–Benson–Bassham (CBB) cycle in NMX2.A1 that is absent in SA-01. Analysis of horizontally transferred gene islands provide evidence that NMX2 selected these genes due to pressure from its HCO3- rich environment, which is in stark contrast to that of the deep subsurface isolated SA-01.



Thursday, September 8 2016 08:30:55 AM

Canopy Temperature and Vegetation Indices from High-Throughput Phenotyping Improve Accuracy of Pedigree and Genomic Selection for Grain Yield in Wheat

Rutkoski, J., Poland, J., Mondal, S., Autrique, E., Perez, L. G., Crossa, J., Reynolds, M., Singh, R.


Genomic selection can be applied prior to phenotyping, enabling shorter breeding cycles and greater rates of genetic gain relative to phenotypic selection. Traits measured using high-throughput phenotyping based on proximal or remote sensing could be useful for improving pedigree and genomic prediction model accuracies for traits not yet possible to phenotype directly. We tested if using aerial measurements of canopy temperature, and green and red normalized difference vegetation index as secondary traits in pedigree and genomic best linear unbiased prediction models could increase accuracy for grain yield in wheat, Triticum aestivum L., using 557 lines in five environments. Secondary traits on training and test sets, and grain yield on the training set were modeled as multivariate, and compared to univariate models with grain yield on the training set only. Cross validation accuracies were estimated within and across-environment, with and without replication, and with and without correcting for days to heading. We observed that, within environment, with unreplicated secondary trait data, and without correcting for days to heading, secondary traits increased accuracies for grain yield by 56% in pedigree, and 70% in genomic prediction models, on average. Secondary traits increased accuracy slightly more when replicated, and considerably less when models corrected for days to heading. In across-environment prediction, trends were similar but less consistent. These results show that secondary traits measured in high-throughput could be used in pedigree and genomic prediction to improve accuracy. This approach could improve selection in wheat during early stages if validated in early-generation breeding plots.



Thursday, September 8 2016 08:30:55 AM

A Split-Ubiquitin Based Strategy Selecting for Protein Complex-Interfering Mutations

Gronemeyer, T., Chollet, J., Werner, S., Glomb, O., Bauerle, A., Johnsson, N.


Understanding the topologies and functions of protein interaction networks requires the selective removal of single interactions. We introduce a selection strategy that enriches among a random library of alleles for mutations that impair the binding to a given partner protein. The selection makes use of a split-ubiquitin based protein interaction assay. This assay provides yeast cells that carry protein complex disturbing mutations with the advantage of being able to survive on uracil-lacking media. Applied to the exemplary interaction between the PB domains of the yeast proteins Bem1 and Cdc24, we performed two independent selections. The selections were either analyzed by Sanger sequencing of isolated clones or by next generation sequencing (NGS) of pools of clones. Both screens enriched for the same mutation in position 833 of Cdc24. Biochemical analysis confirmed that this mutation disturbs the interaction with Bem1 but not the fold of the protein. The larger dataset obtained by NGS achieved a more complete representation of the bipartite interaction interface of Cdc24.



Thursday, September 8 2016 08:30:55 AM

ChloroSeq, an Optimized Chloroplast RNA-Seq Bioinformatic Pipeline, Reveals Remodeling of the Organellar Transcriptome Under Heat Stress

Castandet, B., Hotto, A. M., Strickler, S. R., Stern, D. B.


Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsis thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.



Thursday, September 8 2016 08:30:55 AM

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants

Kwan, E. X., Wang, X. S., Amemiya, H. M., Brewer, B. J., Raghuraman, M. K.


The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae.



Thursday, September 8 2016 08:30:55 AM

Ligand-Bound GeneSwitch Causes Developmental Aberrations in Drosophila that Are Alleviated by the Alternative Oxidase

Andjelković, A., Kemppainen, K. K., Jacobs, H. T.


Culture of Drosophila expressing the steroid-dependent GeneSwitch transcriptional activator under the control of the ubiquitous α-tubulin promoter was found to produce extensive pupal lethality, as well as a range of dysmorphic adult phenotypes, in the presence of high concentrations of the inducing drug RU486. Prominent among these was cleft thorax, seen previously in flies bearing mutant alleles of the nuclear receptor Ultraspiracle and many other mutants, as well as notched wings, leg malformations, and bristle abnormalities. Neither the α-tubulin-GeneSwitch driver nor the inducing drug on their own produced any of these effects. A second GeneSwitch driver, under the control of the daughterless promoter, which gave much lower and more tissue-restricted transgene expression, exhibited only mild bristle abnormalities in the presence of high levels of RU486. Coexpression of the alternative oxidase (AOX) from Ciona intestinalis produced a substantial shift in the developmental outcome toward a wild-type phenotype, which was dependent on the AOX expression level. Neither an enzymatically inactivated variant of AOX, nor GFP, or the alternative NADH dehydrogenase Ndi1 from yeast gave any such rescue. Users of the GeneSwitch system should be aware of the potential confounding effects of its application in developmental studies.



Thursday, September 8 2016 08:30:55 AM

Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

Stroehlein, A. J., Young, N. D., Korhonen, P. K., Chang, B. C. H., Sternberg, P. W., La Rosa, G., Pozio, E., Gasser, R. B.


Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation.



Thursday, September 8 2016 08:30:55 AM

Plethysmography Phenotype QTL in Mice Before and After Allergen Sensitization and Challenge

Kelada, S. N. P.


Allergic asthma is common airway disease that is characterized in part by enhanced airway constriction in response to nonspecific stimuli. Genome-wide association studies have identified multiple loci associated with asthma risk in humans, but these studies have not accounted for gene–environment interactions, which are thought to be important factors in asthma. To identify quantitative trait loci (QTL) that regulate responses to a common human allergen, we applied a house dust mite mouse (HDM) model of allergic airway disease (AAD) to 146 incipient lines of the Collaborative Cross (CC) and the CC founder strains. We employed a longitudinal study design in which mice were phenotyped for response to the bronchoconstrictor methacholine both before and after HDM sensitization and challenge using whole body plethysmography (WBP). There was significant variation in methacholine responsiveness due to both strain and HDM treatment, as reflected by changes in the WBP parameter enhanced pause. We also found that distinct QTL regulate baseline [chromosome (Chr) 18] and post-HDM (Chr 19) methacholine responsiveness and that post-HDM airway responsiveness was correlated with other features of AAD. Finally, using invasive measurements of airway mechanics, we tested whether the Chr 19 QTL affects lung resistance per se using C57BL/6J mice and a consomic strain but found that QTL haplotype did not affect lung resistance. We conclude that aspects of baseline and allergen-induced methacholine responsiveness are associated with genetic variation, and that robust detection of airway resistance QTL in genetically diverse mice will be facilitated by direct measurement of airway mechanics.



Thursday, September 8 2016 08:30:55 AM

Genome-Wide Divergence in the West-African Malaria Vector Anopheles melas

Deitz, K. C., Athrey, G. A., Jawara, M., Overgaard, H. J., Matias, A., Slotman, M. A.


Anopheles melas is a member of the recently diverged An. gambiae species complex, a model for speciation studies, and is a locally important malaria vector along the West-African coast where it breeds in brackish water. A recent population genetic study of An. melas revealed species-level genetic differentiation between three population clusters. An. melas West extends from The Gambia to the village of Tiko, Cameroon. The other mainland cluster, An. melas South, extends from the southern Cameroonian village of Ipono to Angola. Bioko Island, Equatorial Guinea An. melas populations are genetically isolated from mainland populations. To examine how genetic differentiation between these An. melas forms is distributed across their genomes, we conducted a genome-wide analysis of genetic differentiation and selection using whole genome sequencing data of pooled individuals (Pool-seq) from a representative population of each cluster. The An. melas forms exhibit high levels of genetic differentiation throughout their genomes, including the presence of numerous fixed differences between clusters. Although the level of divergence between the clusters is on a par with that of other species within the An. gambiae complex, patterns of genome-wide divergence and diversity do not provide evidence for the presence of pre- and/or postmating isolating mechanisms in the form of speciation islands. These results are consistent with an allopatric divergence process with little or no introgression.



Thursday, September 8 2016 08:30:55 AM

A Genomic Analysis of Factors Driving lincRNA Diversification: Lessons from Plants

Nelson, A. D. L., Forsythe, E. S., Devisetty, U. K., Clausen, D. S., Haug-Batzell, A. K., Meldrum, A. M. R., Frank, M. R., Lyons, E., Beilstein, M. A.


Transcriptomic analyses from across eukaryotes indicate that most of the genome is transcribed at some point in the developmental trajectory of an organism. One class of these transcripts is termed long intergenic noncoding RNAs (lincRNAs). Recently, attention has focused on understanding the evolutionary dynamics of lincRNAs, particularly their conservation within genomes. Here, we take a comparative genomic and phylogenetic approach to uncover factors influencing lincRNA emergence and persistence in the plant family Brassicaceae, to which Arabidopsis thaliana belongs. We searched 10 genomes across the family for evidence of > 5000 lincRNA loci from A. thaliana. From loci conserved in the genomes of multiple species, we built alignments and inferred phylogeny. We then used gene tree/species tree reconciliation to examine the duplication history and timing of emergence of these loci. Emergence of lincRNA loci appears to be linked to local duplication events, but, surprisingly, not whole genome duplication events (WGD), or transposable elements. Interestingly, WGD events are associated with the loss of loci for species having undergone relatively recent polyploidy. Lastly, we identify 1180 loci of the 6480 previously annotated A. thaliana lincRNAs (18%) with elevated levels of conservation. These conserved lincRNAs show higher expression, and are enriched for stress-responsiveness and cis-regulatory motifs known as conserved noncoding sequences (CNSs). These data highlight potential functional pathways and suggest that CNSs may regulate neighboring genes at both the genomic and transcriptomic level. In sum, we provide insight into processes that may influence lincRNA diversification by providing an evolutionary context for previously annotated lincRNAs.



Thursday, September 8 2016 08:30:55 AM

Identification of Genes in Candida glabrata Conferring Altered Responses to Caspofungin, a Cell Wall Synthesis Inhibitor

Rosenwald, A. G., Arora, G., Ferrandino, R., Gerace, E. L., Mohammednetej, M., Nosair, W., Rattila, S., Subic, A. Z., Rolfes, R.


Candida glabrata is an important human fungal pathogen whose incidence continues to rise. Because many clinical isolates are resistant to azole drugs, the drugs of choice to treat such infections are members of the echinocandin family, although there are increasing reports of resistance to these drugs as well. In efforts to better understand the genetic changes that lead to altered responses to echinocandins, we screened a transposon-insertion library of mutants for strains to identify genes that are important for cellular responses to caspofungin, a member of this drug family. We identified 16 genes that, when disrupted, caused increased tolerance, and 48 genes that, when disrupted, caused increased sensitivity compared to the wild-type parental strain. Four of the genes identified as causing sensitivity are orthologs of Saccharomyces cerevisiae genes encoding proteins important for the cell wall integrity (CWI) pathway. In addition, several other genes are orthologs of the high affinity Ca2+ uptake system (HACS) complex genes. We analyzed disruption mutants representing all 64 genes under 33 different conditions, including the presence of cell wall disrupting agents and other drugs, a variety of salts, increased temperature, and altered pH. Further, we generated knockout mutants in different genes within the CWI pathway and the HACS complex, and found that they too exhibited phenotypes consistent with defects in cell wall construction. Our results indicate that small molecules that inhibit the CWI pathway, or that the HACS complex, may be an important means of increasing the efficacy of caspofungin.



Thursday, September 8 2016 08:30:55 AM

From Genotype to Phenotype: Nonsense Variants in SLC13A1 Are Associated with Decreased Serum Sulfate and Increased Serum Aminotransferases

Tise, C. G., Perry, J. A., Anforth, L. E., Pavlovich, M. A., Backman, J. D., Ryan, K. A., Lewis, J. P., OConnell, J. R., Yerges-Armstrong, L. M., Shuldiner, A. R.


Using genomic applications to glean insights into human biology, we systematically searched for nonsense single nucleotide variants (SNVs) that are rare in the general population but enriched in the Old Order Amish (Amish) due to founder effect. We identified two nonlinked, nonsense SNVs (R12X and W48X) in SLC13A1 (allele frequencies 0.29% and 0.74% in the Amish; enriched 1.2-fold and 3.7-fold, compared to the outbred Caucasian population, respectively). SLC13A1 encodes the apical sodium-sulfate cotransporter (NaS1) responsible for sulfate (re)absorption in the kidneys and intestine. SLC13A1 R12X and W48X were independently associated with a 27.6% (P = 2.7 x 10–8) and 27.3% (P = 6.9 x 10–14) decrease in serum sulfate, respectively (P = 8.8 x 10-20 for carriers of either SLC13A1 nonsense SNV). We further performed the first exome- and genome-wide association study (ExWAS/GWAS) of serum sulfate and identified a missense variant (L348P) in SLC26A1, which encodes the basolateral sulfate-anion transporter (Sat1), that was associated with decreased serum sulfate (P = 4.4 x 10–12). Consistent with sulfate’s role in xenobiotic detoxification and protection against acetaminophen-induced hepatotoxicity, SLC13A1 nonsense SNV carriers had higher aminotransferase levels compared to noncarriers. Furthermore, SLC26A1 L348P was associated with lower whole-body bone mineral density (BMD) and higher serum calcium, consistent with the osteochondrodysplasia exhibited by dogs and sheep with naturally occurring, homozygous, loss-of-function mutations in Slc13a1. This study demonstrates the power and translational potential of systematic identification and characterization of rare, loss-of-function variants and warrants additional studies to better understand the importance of sulfate in human physiology, disease, and drug toxicity.



Thursday, September 8 2016 08:30:55 AM

Optimizing Training Population Data and Validation of Genomic Selection for Economic Traits in Soft Winter Wheat

Hoffstetter, A., Cabrera, A., Huang, M., Sneller, C.


Genomic selection (GS) is a breeding tool that estimates breeding values (GEBVs) of individuals based solely on marker data by using a model built using phenotypic and marker data from a training population (TP). The effectiveness of GS increases as the correlation of GEBVs and phenotypes (accuracy) increases. Using phenotypic and genotypic data from a TP of 470 soft winter wheat lines, we assessed the accuracy of GS for grain yield, Fusarium Head Blight (FHB) resistance, softness equivalence (SE), and flour yield (FY). Four TP data sampling schemes were tested: (1) use all TP data, (2) use subsets of TP lines with low genotype-by-environment interaction, (3) use subsets of markers significantly associated with quantitative trait loci (QTL), and (4) a combination of 2 and 3. We also correlated the phenotypes of relatives of the TP to their GEBVs calculated from TP data. The GS accuracy within the TP using all TP data ranged from 0.35 (FHB) to 0.62 (FY). On average, the accuracy of GS from using subsets of data increased by 54% relative to using all TP data. Using subsets of markers selected for significant association with the target trait had the greatest impact on GS accuracy. Between-environment prediction accuracy was also increased by using data subsets. The accuracy of GS when predicting the phenotypes of TP relatives ranged from 0.00 to 0.85. These results suggest that GS could be useful for these traits and GS accuracy can be greatly improved by using subsets of TP data.



Thursday, September 8 2016 08:30:55 AM

Genome-Wide Association Mapping for Female Infertility in Inbred Mice

Liu, J.-L., Wang, T.-S., Zhao, M.


The genetic factors underlying female infertility in humans are only partially understood. Here, we performed a genome-wide association study of female infertility in 25 inbred mouse strains by using publicly available SNP data. As a result, a total of four SNPs were identified after chromosome-wise multiple test correction. The first SNP rs29972765 is located in a gene desert on chromosome 18, about 72 kb upstream of Skor2 (SKI family transcriptional corepressor 2). The second SNP rs30415957 resides in the intron of Plce1 (phospholipase C epsilon 1). The remaining two SNPs (rs30768258 and rs31216810) are close to each other on chromosome 19, in the vicinity of Sorbs1 (sorbin and SH3 domain containing 1). Using quantitative RT-PCR, we found that Sorbs1 is highly expressed in the mouse uterus during embryo implantation. Knockdown of Sorbs1 by siRNA attenuates the induction of differentiation marker gene Prl8a2 (decidual prolactin-related protein) in an in vitro model of decidualization using mouse endometrial stromal cells, suggesting that Sorbs1 may be a potential candidate gene for female infertility in mice. Our results may represent an opportunity to further understand female infertility in humans.



Thursday, September 8 2016 08:30:55 AM

Inter-genomic DNA Exchanges and Homeologous Gene Silencing Shaped the Nascent Allopolyploid Coffee Genome (Coffea arabica L.)

Lashermes, P., Hueber, Y., Combes, M.-C., Severac, D., Dereeper, A.


Allopolyploidization is a biological process that has played a major role in plant speciation and evolution. Genomic changes are common consequences of polyploidization, but their dynamics over time are still poorly understood. Coffea arabica, a recently formed allotetraploid, was chosen to study genetic changes that accompany allopolyploid formation. Both RNA-seq and DNA-seq data were generated from two genetically distant C. arabica accessions. Genomic structural variation was investigated using C. canephora, one of its diploid progenitors, as reference genome. The fate of 9047 duplicate homeologous genes was inferred and compared between the accessions. The pattern of SNP density along the reference genome was consistent with the allopolyploid structure. Large genomic duplications or deletions were not detected. Two homeologous copies were retained and expressed in 96% of the genes analyzed. Nevertheless, duplicated genes were found to be affected by various genomic changes leading to homeolog loss or silencing. Genetic and epigenetic changes were evidenced that could have played a major role in the stabilization of the unique ancestral allotetraploid and its subsequent diversification. While the early evolution of C. arabica mainly involved homeologous crossover exchanges, the later stage appears to have relied on more gradual evolution involving gene conversion and homeolog silencing.



Thursday, September 8 2016 08:30:55 AM

A Splice Defect in the EDA Gene in Dogs with an X-Linked Hypohidrotic Ectodermal Dysplasia (XLHED) Phenotype

Waluk, D. P., Zur, G., Kaufmann, R., Welle, M. M., Jagannathan, V., Drogemuller, C., Muller, E. J., Leeb, T., Galichet, A.


X-linked hypohidrotic ectodermal dysplasia (XLHED) caused by variants in the EDA gene represents the most common ectodermal dysplasia in humans. We investigated three male mixed-breed dogs with an ectodermal dysplasia phenotype characterized by marked hypotrichosis and multifocal complete alopecia, almost complete absence of sweat and sebaceous glands, and altered dentition with missing and abnormally shaped teeth. Analysis of SNP chip genotypes and whole genome sequence data from the three affected dogs revealed that the affected dogs shared the same haplotype on a large segment of the X-chromosome, including the EDA gene. Unexpectedly, the whole genome sequence data did not reveal any nonsynonymous EDA variant in the affected dogs. We therefore performed an RNA-seq experiment on skin biopsies to search for changes in the transcriptome. This analysis revealed that the EDA transcript in the affected dogs lacked 103 nucleotides encoded by exon 2. We speculate that this exon skipping is caused by a genetic variant located in one of the large introns flanking this exon, which was missed by whole genome sequencing with the illumina short read technology. The altered EDA transcript splicing most likely causes the observed ectodermal dysplasia in the affected dogs. These dogs thus offer an excellent opportunity to gain insights into the complex splicing processes required for expression of the EDA gene, and other genes with large introns.



Thursday, September 8 2016 08:30:56 AM

Sensitivity of Allelic Divergence to Genomic Position: Lessons from the Drosophila tan Gene

John, A. V., Sramkoski, L. L., Walker, E. A., Cooley, A. M., Wittkopp, P. J.


To identify genetic variants underlying changes in phenotypes within and between species, researchers often utilize transgenic animals to compare the function of alleles in different genetic backgrounds. In Drosophila, targeted integration mediated by the C31 integrase allows activity of alternative alleles to be compared at the same genomic location. By using the same insertion site for each transgene, position effects are generally assumed to be controlled for because both alleles are surrounded by the same genomic context. Here, we test this assumption by comparing the activity of tan alleles from two Drosophila species, D. americana and D. novamexicana, at five different genomic locations in D. melanogaster. We found that the relative effects of these alleles varied among insertion sites, with no difference in activity observed between them at two sites. One of these sites simply silenced both transgenes, but the other allowed expression of both alleles that was sufficient to rescue a mutant phenotype yet failed to reveal the functional differences between the two alleles. These results suggest that more than one insertion site should be used when comparing the activity of transgenes because failing to do so could cause functional differences between alleles to go undetected.



Thursday, September 8 2016 08:30:56 AM

An Intronic MBTPS2 Variant Results in a Splicing Defect in Horses with Brindle Coat Texture

Murgiano, L., Waluk, D. P., Towers, R., Wiedemar, N., Dietrich, J., Jagannathan, V., Drogemuller, M., Balmer, P., Druet, T., Galichet, A., Penedo, M. C., Muller, E. J., Roosje, P., Welle, M. M., Leeb, T.


We investigated a family of horses exhibiting irregular vertical stripes in their hair coat texture along the neck, back, hindquarters, and upper legs. This phenotype is termed "brindle" by horse breeders. We propose the term "brindle 1 (BR1)" for this specific form of brindle. In some BR1 horses, the stripes were also differentially pigmented. Pedigree analyses were suggestive of a monogenic X-chromosomal semidominant mode of inheritance. Haplotype analyses identified a 5 Mb candidate region on chromosome X. Whole genome sequencing of four BR1 and 60 nonbrindle horses identified 61 private variants in the critical interval, none of them located in an exon of an annotated gene. However, one of the private variants was close to an exon/intron boundary in intron 10 of the MBTPS2 gene encoding the membrane bound transcription factor peptidase, site 2 (c.1437+4T>C). Different coding variants in this gene lead to three related genodermatoses in human patients. We therefore analyzed MBTPS2 transcripts in skin, and identified an aberrant transcript in a BR1 horse, which lacked the entire exon 10 and parts of exon 11. The MBTPS2:c1437+4T>C variant showed perfect cosegregation with the brindle phenotype in the investigated family, and was absent from 457 control horses of diverse breeds. Altogether, our genetic data, and previous knowledge on MBTPS2 function in the skin, suggest that the identified MBTPS2 intronic variant leads to partial exon skipping, and causes the BR1 phenotype in horses.



Thursday, September 8 2016 08:30:56 AM

Evidence for Regulation of ECM3 Expression by Methylation of Histone H3 Lysine 4 and Intergenic Transcription in Saccharomyces cerevisiae

Raupach, E. A., Martens, J. A., Arndt, K. M.


Transcription of nonprotein-coding DNA is widespread in eukaryotes and plays important regulatory roles for many genes, including genes that are misregulated in cancer cells. Its pervasiveness presents the potential for a wealth of diverse regulatory roles for noncoding transcription. We previously showed that the act of transcribing noncoding DNA (ncDNA) across the promoter of the protein-coding SER3 gene in Saccharomyces cerevisiae positions nucleosomes over the upstream activating sequences, leading to strong repression of SER3 transcription. To explore the possibility of other regulatory roles for ncDNA transcription, we selected six candidate S. cerevisiae genes that express ncRNAs over their promoters and analyzed the regulation of one of these genes, ECM3, in detail. Because noncoding transcription can lead to changes in the local chromatin landscape that impinge on the expression of nearby coding genes, we surveyed the effects of various chromatin regulators on the expression of ECM3. These analyses identified roles for the Paf1 complex in positively regulating ECM3 transcription through methylation of histone H3 at lysine 4 (K4) and for Paf1 in controlling the pattern of intergenic transcription at this locus. By deleting a putative promoter for the noncoding transcription unit that lies upstream of ECM3, we provide evidence for a positive correlation between intergenic transcription and ECM3 expression. Our results are consistent with a model in which cotranscriptional methylation of histone H3 K4, mediated by the Paf1 complex and noncoding transcription, leads to activation of ECM3 transcription.



Thursday, September 8 2016 08:30:56 AM

Aspergillus fumigatus MADS-Box Transcription Factor rlmA Is Required for Regulation of the Cell Wall Integrity and Virulence

Rocha, M. C., Fabri, J. H. T. M., Franco de Godoy, K., Alves de Castro, P., Hori, J. I., Ferreira da Cunha, A., Arentshorst, M., Ram, A. F. J., van den Hondel, C. A. M. J. J., Goldman, G. H., Malavazi, I.


The Cell Wall Integrity (CWI) pathway is the primary signaling cascade that controls the de novo synthesis of the fungal cell wall, and in Saccharomyces cerevisiae this event is highly dependent on the RLM1 transcription factor. Here, we investigated the function of RlmA in the fungal pathogen Aspergillus fumigatus. We show that the rlmA strain exhibits an altered cell wall organization in addition to defects related to vegetative growth and tolerance to cell wall-perturbing agents. A genetic analysis indicated that rlmA is positioned downstream of the pkcA and mpkA genes in the CWI pathway. As a consequence, rlmA loss-of-function leads to the altered expression of genes encoding cell wall-related proteins. RlmA positively regulates the phosphorylation of MpkA and is induced at both protein and transcriptional levels during cell wall stress. The rlmA was also involved in tolerance to oxidative damage and transcriptional regulation of genes related to oxidative stress adaptation. Moreover, the rlmA strain had attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Our results suggest that RlmA functions as a transcription factor in the A. fumigatus CWI pathway, acting downstream of PkcA-MpkA signaling and contributing to the virulence of this fungus.



Thursday, September 8 2016 08:30:56 AM

Scan-o-matic: High-Resolution Microbial Phenomics at a Massive Scale

Zackrisson, M., Hallin, J., Ottosson, L.-G., Dahl, P., Fernandez-Parada, E., Landstrom, E., Fernandez-Ricaud, L., Kaferle, P., Skyman, A., Stenberg, S., Omholt, S., Petrovič, U., Warringer, J., Blomberg, A.


The capacity to map traits over large cohorts of individuals—phenomics—lags far behind the explosive development in genomics. For microbes, the estimation of growth is the key phenotype because of its link to fitness. We introduce an automated microbial phenomics framework that delivers accurate, precise, and highly resolved growth phenotypes at an unprecedented scale. Advancements were achieved through the introduction of transmissive scanning hardware and software technology, frequent acquisition of exact colony population size measurements, extraction of population growth rates from growth curves, and removal of spatial bias by reference-surface normalization. Our prototype arrangement automatically records and analyzes close to 100,000 growth curves in parallel. We demonstrate the power of the approach by extending and nuancing the known salt-defense biology in baker’s yeast. The introduced framework represents a major advance in microbial phenomics by providing high-quality data for extensive cohorts of individuals and generating well-populated and standardized phenomics databases