GENETICS 2014 Web Spotlights G3: Genes|Genomes|Genetics 2014 Web Spotlights The GSA Reporter GSA e-News Genes to Genomes: the GSA blog Advertising with GSA Spotlight on Undergraduate Research

 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       What's Inside the Current Issue of G3    Tuesday, May 3 2016 08:37:05 AM The Genetic Basis of Baculum Size and Shape Variation in Mice Schultz, N. G., Ingels, J., Hillhouse, A., Wardwell, K., Chang, P. L., Cheverud, J. M., Lutz, C., Lu, L., Williams, R. W., Dean, M. D. The rapid divergence of male genitalia is a preeminent evolutionary pattern. This rapid divergence is especially striking in the baculum, a bone that occurs in the penis of many mammalian species. Closely related species often display diverse baculum morphology where no other morphological differences can be discerned. While this fundamental pattern of evolution has been appreciated at the level of gross morphology, nearly nothing is known about the genetic basis of size and shape divergence. Quantifying the genetic basis of baculum size and shape variation has been difficult because these structures generally lack obvious landmarks, so comparing them in three dimensions is not straightforward. Here, we develop a novel morphometric approach to quantify size and shape variation from three-dimensional micro-CT scans taken from 369 bacula, representing 75 distinct strains of the BXD family of mice. We identify two quantitative trait loci (QTL) that explain ~50% of the variance in baculum size, and a third QTL that explains more than 20% of the variance in shape. Together, our study demonstrates that baculum morphology may diverge relatively easily, with mutations at a few loci of large effect that independently modulate size and shape. Based on a combination of bioinformatic investigations and new data on RNA expression, we prioritized these QTL to 16 candidate genes, which have hypothesized roles in bone morphogenesis and may enable future genetic manipulation of baculum morphology. Tuesday, May 3 2016 08:37:05 AM Does 3D Phenotyping Yield Substantial Insights in the Genetics of the Mouse Mandible Shape? Navarro, N., Maga, A. M. We describe the application of high-resolution 3D microcomputed tomography, together with 3D landmarks and geometric morphometrics, to validate and further improve previous quantitative genetic studies that reported QTL responsible for variation in the mandible shape of laboratory mice using a new backcross between C57BL/6J and A/J inbred strains. Despite the increasing availability of 3D imaging techniques, artificial flattening of the mandible by 2D imaging techniques seems at first an acceptable compromise for large-scale phenotyping protocols, thanks to an abundance of low-cost digital imaging systems such as microscopes or digital cameras. We evaluated the gain of information from considering explicitly this additional third dimension, and also from capturing variation on the bone surface where no precise anatomical landmark can be marked. Multivariate QTL mapping conducted with different landmark configurations (2D vs. 3D; manual vs. semilandmarks) broadly agreed with the findings of previous studies. Significantly more QTL (23) were identified and more precisely mapped when the mandible shape was captured with a large set of semilandmarks coupled with manual landmarks. It appears that finer phenotypic characterization of the mandibular shape with 3D landmarks, along with higher density genotyping, yields better insights into the genetic architecture of mandibular development. Most of the main variation is, nonetheless, preferentially embedded in the natural 2D plane of the hemi-mandible, reinforcing the results of earlier influential investigations. Tuesday, May 3 2016 08:37:05 AM Genomic Bayesian Prediction Model for Count Data with Genotype x Environment Interaction Montesinos-Lopez, A., Montesinos-Lopez, O. A., Crossa, J., Burgueno, J., Eskridge, K. M., Falconi-Castillo, E., He, X., Singh, P., Cichy, K. Genomic tools allow the study of the whole genome, and facilitate the study of genotype-environment combinations and their relationship with phenotype. However, most genomic prediction models developed so far are appropriate for Gaussian phenotypes. For this reason, appropriate genomic prediction models are needed for count data, since the conventional regression models used on count data with a large sample size ($${\mathit{n}}_{\mathit{T}}$$) and a small number of parameters (p) cannot be used for genomic-enabled prediction where the number of parameters (p) is larger than the sample size ($${\mathit{n}}_{\mathit{T}}$$). Here, we propose a Bayesian mixed-negative binomial (BMNB) genomic regression model for counts that takes into account genotype by environment $$\left(\mathit{G\times E}\right)$$ interaction. We also provide all the full conditional distributions to implement a Gibbs sampler. We evaluated the proposed model using a simulated data set, and a real wheat data set from the International Maize and Wheat Improvement Center (CIMMYT) and collaborators. Results indicate that our BMNB model provides a viable option for analyzing count data. Tuesday, May 3 2016 08:37:05 AM Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes Hamaji, T., Mogi, Y., Ferris, P. J., Mori, T., Miyagishima, S., Kabeya, Y., Nishimura, Y., Toyoda, A., Noguchi, H., Fujiyama, A., Olson, B. J. S. C., Marriage, T. N., Nishii, I., Umen, J. G., Nozaki, H. Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii. Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii. Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT–, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale–V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale. These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs. Tuesday, May 3 2016 08:37:05 AM Comparative and Transcriptome Analyses Uncover Key Aspects of Coding- and Long Noncoding RNAs in Flatworm Mitochondrial Genomes Ross, E., Blair, D., Guerrero-Hernandez, C., Alvarado, A. S. Exploiting the conservation of various features of mitochondrial genomes has been instrumental in resolving phylogenetic relationships. Despite extensive sequence evidence, it has not previously been possible to conclusively resolve some key aspects of flatworm mitochondrial genomes, including generally conserved traits, such as start codons, noncoding regions, the full complement of tRNAs, and whether ATP8 is, or is not, encoded by this extranuclear genome. In an effort to address these difficulties, we sought to determine the mitochondrial transcriptomes and genomes of sexual and asexual taxa of freshwater triclads, a group previously poorly represented in flatworm mitogenomic studies. We have discovered evidence for an alternative start codon, an extended cox1 gene, a previously undescribed conserved open reading frame, long noncoding RNAs, and a highly conserved gene order across the large evolutionary distances represented within the triclads. Our findings contribute to the expansion and refinement of mitogenomics to address evolutionary issues in this diverse group of animals. Tuesday, May 3 2016 08:37:05 AM Massive Amplification at an Unselected Locus Accompanies Complex Chromosomal Rearrangements in Yeast Thierry, A., Khanna, V., Dujon, B. Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them with specific phenotypic adaptations via increased expression levels. However, the presence of multiple gene copies in natural genomes has generally not been found in the absence of specific functional selection. Here, we show that the massive amplification of a chromosomal locus (up to 880 copies per cell) occurs in the absence of any direct selection, and is associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained from mutants with restored phenotypes that spontaneously appeared from genetically engineered strains of the yeast Saccharomyces cerevisiae suffering from severe fitness reduction. Grossly extended chromosomes (macrotene) were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints, and comparisons between mutants, revealed novel mechanisms causing their formation, whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and their consequences. Tuesday, May 3 2016 08:37:05 AM The Genetic Linkage Map of the Medicinal Mushroom Agaricus subrufescens Reveals Highly Conserved Macrosynteny with the Congeneric Species Agaricus bisporus Foulongne-Oriol, M., Rocha de Brito, M., Cabannes, D., Clement, A., Spataro, C., Moinard, M., Dias, E. S., Callac, P., Savoie, J.-M. Comparative linkage mapping can rapidly facilitate the transfer of genetic information from model species to orphan species. This macrosynteny analysis approach has been extensively used in plant species, but few example are available in fungi, and even fewer in mushroom crop species. Among the latter, the Agaricus genus comprises the most cultivable or potentially cultivable species. Agaricus bisporus, the button mushroom, is the model for edible and cultivable mushrooms. We have developed the first genetic linkage map for the basidiomycete A. subrufescens, an emerging mushroom crop known for its therapeutic properties and potential medicinal applications. The map includes 202 markers distributed over 16 linkage groups (LG), and covers a total length of 1701 cM, with an average marker spacing of 8.2 cM. Using 96 homologous loci, we also demonstrated the high level of macrosynteny with the genome of A. bisporus. The 13 main LG of A. subrufescens were syntenic to the 13 A. bisporus chromosomes. A disrupted synteny was observed for the three remaining A. subrufescens LG. Electronic mapping of a collection of A. subrufescens expressed sequence tags on A. bisporus genome showed that the homologous loci were evenly spread, with the exception of a few local hot or cold spots of homology. Our results were discussed in the light of Agaricus species evolution process. The map provides a framework for future genetic or genomic studies of the medicinal mushroom A. subrufescens. Tuesday, May 3 2016 08:37:05 AM Staufen Negatively Modulates MicroRNA Activity in Caenorhabditis elegans Ren, Z., Veksler-Lublinsky, I., Morrissey, D., Ambros, V. The double-stranded RNA-binding protein Staufen has been implicated in various posttranscriptional gene regulatory processes. Here, we demonstrate that the Caenorhabditis elegans homolog of Staufen, STAU-1, functionally interacts with microRNAs. Loss-of-function mutations of stau-1 significantly suppress phenotypes of let-7 family microRNA mutants, a hypomorphic allele of dicer, and a lsy-6 microRNA partial loss-of-function mutant. Furthermore, STAU-1 modulates the activity of lin-14, a target of lin-4 and let-7 family microRNAs, and this modulation is abolished when the 3' untranslated region of lin-14 is removed. Deep sequencing of small RNA cDNA libraries reveals no dramatic change in the levels of microRNAs or other small RNA populations between wild-type and stau-1 mutants, with the exception of certain endogenous siRNAs in the WAGO pathway. The modulation of microRNA activity by STAU-1 does not seem to be associated with the previously reported enhanced exogenous RNAi (Eri) phenotype of stau-1 mutants, since eri-1 exhibits the opposite effect on microRNA activity. Altogether, our results suggest that STAU-1 negatively modulates microRNA activity downstream of microRNA biogenesis, possibly by competing with microRNAs for binding on the 3' untranslated region of target mRNAs. Tuesday, May 3 2016 08:37:05 AM Comparative Transcriptomics Indicates a Role for SHORT VEGETATIVE PHASE (SVP) Genes in Mimulus guttatus Vernalization Response Preston, J. C., Zhong, J., McKeown, M., den Bakker, M., Friedman, J. The timing of reproduction in response to variable environmental conditions is critical to plant fitness, and is a major driver of taxon differentiation. In the yellow monkey flower, Mimulus guttatus, geographically distinct North American populations vary in their photoperiod and chilling (vernalization) requirements for flowering, suggesting strong local adaptation to their surroundings. Previous analyses revealed quantitative trait loci (QTL) underlying short-day mediated vernalization responsiveness using two annual M. guttatus populations that differed in their vernalization response. To narrow down candidate genes responsible for this variation, and to reveal potential downstream genes, we conducted comparative transcriptomics and quantitative PCR (qPCR) in shoot apices of parental vernalization responsive IM62, and unresponsive LMC24 inbred lines grown under different photoperiods and temperatures. Our study identified several metabolic, hormone signaling, photosynthetic, stress response, and flowering time genes that are differentially expressed between treatments, suggesting a role for their protein products in short-day-mediated vernalization responsiveness. Only a small subset of these genes intersected with candidate genes from the previous QTL study, and, of the main candidates tested with qPCR under nonpermissive conditions, only SHORT VEGETATIVE PHASE (SVP) gene expression met predictions for a population-specific short-day-repressor of flowering that is repressed by cold. Tuesday, May 3 2016 08:37:05 AM Latitudinal Clines of the Human Vitamin D Receptor and Skin Color Genes Tiosano, D., Audi, L., Climer, S., Zhang, W., Templeton, A. R., Fernandez-Cancio, M., Gershoni-Baruch, R., Sanchez-Muro, J. M., El Kholy, M., Hochberg, Z. The well-documented latitudinal clines of genes affecting human skin color presumably arise from the need for protection from intense ultraviolet radiation (UVR) vs. the need to use UVR for vitamin D synthesis. Sampling 751 subjects from a broad range of latitudes and skin colors, we investigated possible multilocus correlated adaptation of skin color genes with the vitamin D receptor gene (VDR), using a vector correlation metric and network method called BlocBuster. We discovered two multilocus networks involving VDR promoter and skin color genes that display strong latitudinal clines as multilocus networks, even though many of their single gene components do not. Considered one by one, the VDR components of these networks show diverse patterns: no cline, a weak declining latitudinal cline outside of Africa, and a strong in- vs. out-of-Africa frequency pattern. We confirmed these results with independent data from HapMap. Standard linkage disequilibrium analyses did not detect these networks. We applied BlocBuster across the entire genome, showing that our networks are significant outliers for interchromosomal disequilibrium that overlap with environmental variation relevant to the genes’ functions. These results suggest that these multilocus correlations most likely arose from a combination of parallel selective responses to a common environmental variable and coadaptation, given the known Mendelian epistasis among VDR and the skin color genes. Tuesday, May 3 2016 08:37:05 AM Transcriptomic Analysis Identifies Candidate Genes and Gene Sets Controlling the Response of Porcine Peripheral Blood Mononuclear Cells to Poly I:C Stimulation Wang, J., Wang, Y., Wang, H., Wang, H., Liu, J.-F., Wu, Y., Guo, J. Polyinosinic-polycytidylic acid (poly I:C), a synthetic dsRNA analog, has been demonstrated to have stimulatory effects similar to viral dsRNA. To gain deep knowledge of the host transcriptional response of pigs to poly I:C stimulation, in the present study, we cultured and stimulated peripheral blood mononuclear cells (PBMC) of piglets of one Chinese indigenous breed (Dapulian) and one modern commercial breed (Landrace) with poly I:C, and compared their transcriptional profiling using RNA-sequencing (RNA-seq). Our results indicated that poly I:C stimulation can elicit significantly differentially expressed (DE) genes in Dapulian (g = 290) as well as Landrace (g = 85). We also performed gene set analysis using the Gene Set Enrichment Analysis (GSEA) package, and identified some significantly enriched gene sets in Dapulian (g = 18) and Landrace (g = 21). Most of the shared DE genes and gene sets were immune-related, and may play crucial rules in the immune response of poly I:C stimulation. In addition, we detected large sets of significantly DE genes and enriched gene sets when comparing the gene expression profile between the two breeds, including control and poly I:C stimulation groups. Besides immune-related functions, some of the DE genes and gene sets between the two breeds were involved in development and growth of various tissues, which may be correlated with the different characteristics of the two breeds. The DE genes and gene sets detected herein provide crucial information towards understanding the immune regulation of antiviral responses, and the molecular mechanisms of different genetic resistance to viral infection, in modern and indigenous pigs. Tuesday, May 3 2016 08:37:05 AM Chicken gga-miR-103-3p Targets CCNE1 and TFDP2 and Inhibits MDCC-MSB1 Cell Migration Han, B., Lian, L., Li, X., Zhao, C., Qu, L., Liu, C., Song, J., Yang, N. Marek’s disease (MD) is a highly contagious viral neoplastic disease caused by Marek’s disease virus (MDV), which can lead to huge economic losses in the poultry industry. Recently, microRNAs (miRNAs) have been found in various cancers and tumors. In recent years, 994 mature miRNAs have been identified through deep sequencing in chickens, but only a few miRNAs have been investigated further in terms of their function. Previously, gga-miR-103-3p was found downregulated in MDV-infected samples by using Solexa deep sequencing. In this study, we further verified the expression of gga-miR-103-3p among MDV-infected spleen, MD lymphoma from liver, noninfected spleen, and noninfected liver, by qPCR. The results showed that the expression of gga-miR-103-3p was decreased in MDV-infected tissues, which was consistent with our previous study. Furthermore, two target genes of gga-miR-103-3p, cyclin E1 (CCNE1) and transcription factor Dp-2 (E2F dimerization partner 2) (TFDP2), were predicted and validated by luciferase reporter assay, qPCR, and western blot analysis. The results suggested that CCNE1 and TFDP2 are direct targets of gga-miR-103-3p in chickens. Subsequent cell proliferation and migration assay showed that gga-miR-103-3p suppressed MDCC-MSB1 migration, but did not obviously modulate MDCC-MSB1 cell proliferation. In conclusion, gga-miR-103-3p targets the CCNE1 and TFDP2 genes, and suppresses cell migration, which indicates that it might play an important role in MD tumor transformation. Tuesday, May 3 2016 08:37:05 AM Conflation of Short Identity-by-Descent Segments Bias Their Inferred Length Distribution Chiang, C. W. K., Ralph, P., Novembre, J. Identity-by-descent (IBD) is a fundamental concept in genetics with many applications. In a common definition, two haplotypes are said to share an IBD segment if that segment is inherited from a recent shared common ancestor without intervening recombination. Segments several cM long can be efficiently detected by a number of algorithms using high-density SNP array data from a population sample, and there are currently efforts to detect shorter segments from sequencing. Here, we study a problem of identifiability: because existing approaches detect IBD based on contiguous segments of identity-by-state, inferred long segments of IBD may arise from the conflation of smaller, nearby IBD segments. We quantified this effect using coalescent simulations, finding that significant proportions of inferred segments 1–2 cM long are results of conflations of two or more shorter segments, each at least 0.2 cM or longer, under demographic scenarios typical for modern humans for all programs tested. The impact of such conflation is much smaller for longer (> 2 cM) segments. This biases the inferred IBD segment length distribution, and so can affect downstream inferences that depend on the assumption that each segment of IBD derives from a single common ancestor. As an example, we present and analyze an estimator of the de novo mutation rate using IBD segments, and demonstrate that unmodeled conflation leads to underestimates of the ages of the common ancestors on these segments, and hence a significant overestimate of the mutation rate. Understanding the conflation effect in detail will make its correction in future methods more tractable. Tuesday, May 3 2016 08:37:05 AM Mapping Challenging Mutations by Whole-Genome Sequencing Smith, H. E., Fabritius, A. S., Jaramillo-Lambert, A., Golden, A. Whole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homozygous variants). However, such approaches are not entirely suitable for the characterization of a variety of more challenging mutations, such as dominant and semidominant alleles or multigenic traits. Therefore, we have developed strategies for the identification of those classes of mutations, using polymorphism mapping in Caenorhabditis elegans as our model for validation. We also report an alternative approach for mutation identification from traditional recombinant crosses, and a solution to the technical challenge of sequencing sterile or terminally arrested strains where population size is limiting. The methods described herein extend the applicability of whole-genome sequencing to a broader spectrum of mutations, including classes that are difficult to map by traditional means. Tuesday, May 3 2016 08:37:05 AM Characterization of Chicken MMP13 Expression and Genetic Effect on Egg Production Traits of Its Promoter Polymorphisms Yuan, Z., Chen, Y., Chen, Q., Guo, M., Kang, L., Zhu, G., Jiang, Y. Extracelluar matrix undergoes constant remodeling, cell–cell, and cell–matrix interactions during chicken ovarian follicle growth, which is coordinated by matrix metalloproteinases (MMPs), and their associated endogenous inhibitors (TIMPs). Transcriptome analysis revealed upregulation of MMP13 in sexually mature chicken ovaries. In this study, we found that the expression of MMP13 in chicken ovary was stably elevated from 60 d to 159 d, and was significantly higher at 159 d than at the other three developmental stages (P < 0.05). The expression of MMP13 mRNA increased from SW (small white follicles) to F5 (fifth largest follicles), then decreased to F1 (first largest follicles), and dramatically increased again in POF1 (newly postovulatory follicles) follicles (P < 0.05). The MMP13 protein was localized in stroma cells and primordial follicles of sexually immature chicken ovaries, in the theca cell layers of all sized follicles of sexually mature chicken ovaries. Furthermore, we identified a positive element (positions –1863 to –1036) controlling chicken MMP13 transcription, and, in this region, six single nucleotide polymorphisms were found and genotyped in chicken populations. In the White Recessive Rock population, hens with A–1356-C–1079/A–1356-C–1079 genotype had earlier "age at first laying" than those with G–1356-T–1079/G–1356-T–1079 genotype (P < 0.05), and exhibited significantly lower transcriptional activity (P < 0.01). Collectively, chicken MMP13 plays an important role in ovarian follicle growth and regression, and polymorphisms in its promoter region could be used as molecular markers for improving the trait "age at first laying" in chicken breeding. Tuesday, May 3 2016 08:37:05 AM Genomic Selection in Multi-environment Crop Trials Oakey, H., Cullis, B., Thompson, R., Comadran, J., Halpin, C., Waugh, R. Genomic selection in crop breeding introduces modeling challenges not found in animal studies. These include the need to accommodate replicate plants for each line, consider spatial variation in field trials, address line by environment interactions, and capture nonadditive effects. Here, we propose a flexible single-stage genomic selection approach that resolves these issues. Our linear mixed model incorporates spatial variation through environment-specific terms, and also randomization-based design terms. It considers marker, and marker by environment interactions using ridge regression best linear unbiased prediction to extend genomic selection to multiple environments. Since the approach uses the raw data from line replicates, the line genetic variation is partitioned into marker and nonmarker residual genetic variation (i.e., additive and nonadditive effects). This results in a more precise estimate of marker genetic effects. Using barley height data from trials, in 2 different years, of up to 477 cultivars, we demonstrate that our new genomic selection model improves predictions compared to current models. Analyzing single trials revealed improvements in predictive ability of up to 5.7%. For the multiple environment trial (MET) model, combining both year trials improved predictive ability up to 11.4% compared to a single environment analysis. Benefits were significant even when fewer markers were used. Compared to a single-year standard model run with 3490 markers, our partitioned MET model achieved the same predictive ability using between 500 and 1000 markers depending on the trial. Our approach can be used to increase accuracy and confidence in the selection of the best lines for breeding and/or, to reduce costs by using fewer markers. Tuesday, May 3 2016 08:37:05 AM The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism Freitas, F. Z., Virgilio, S., Cupertino, F. B., Kowbel, D. J., Fioramonte, M., Gozzo, F. C., Glass, N. L., Bertolini, M. C. When exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. In this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the Neurospora crassa SEB-1 transcription factor that binds to the Stress Response Element (STRE) under heat stress. Orthologs of SEB-1 have been functionally characterized in a few filamentous fungi as being involved in stress responses; however, the molecular mechanisms mediated by this transcription factor may not be conserved. Here, we provide evidences for the involvement of N. crassa SEB-1 in multiple cellular processes, including response to heat, as well as osmotic and oxidative stress. The seb-1 strain displayed reduced growth under these conditions, and genes encoding stress-responsive proteins were differentially regulated in the seb-1 strain grown under the same conditions. In addition, the SEB-1-GFP protein translocated from the cytosol to the nucleus under heat, osmotic, and oxidative stress conditions. SEB-1 also regulates the metabolism of the reserve carbohydrates glycogen and trehalose under heat stress, suggesting an interconnection between metabolism control and this environmental condition. We demonstrated that SEB-1 binds in vivo to the promoters of genes encoding glycogen metabolism enzymes and regulates their expression. A genome-wide transcriptional profile of the seb-1 strain under heat stress was determined by RNA-seq, and a broad range of cellular processes was identified that suggests a role for SEB-1 as a protein interconnecting these mechanisms. Tuesday, May 3 2016 08:37:05 AM Patterns of Transcriptional Response to 1,25-Dihydroxyvitamin D3 and Bacterial Lipopolysaccharide in Primary Human Monocytes Kariuki, S. N., Blischak, J. D., Nakagome, S., Witonsky, D. B., Di Rienzo, A. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), plays an important immunomodulatory role, regulating transcription of genes in the innate and adaptive immune system. The present study examines patterns of transcriptome-wide response to 1,25D, and the bacterial lipopolysaccharide (LPS) in primary human monocytes, to elucidate pathways underlying the effects of 1,25D on the immune system. Monocytes obtained from healthy individuals of African-American and European-American ancestry were treated with 1,25D, LPS, or both, simultaneously. The addition of 1,25D during stimulation with LPS induced significant upregulation of genes in the antimicrobial and autophagy pathways, and downregulation of proinflammatory response genes compared to LPS treatment alone. A joint Bayesian analysis enabled clustering of genes into patterns of shared transcriptional response across treatments. The biological pathways enriched within these expression patterns highlighted several mechanisms through which 1,25D could exert its immunomodulatory role. Pathways such as mTOR signaling, EIF2 signaling, IL-8 signaling, and Tec Kinase signaling were enriched among genes with opposite transcriptional responses to 1,25D and LPS, respectively, highlighting the important roles of these pathways in mediating the immunomodulatory activity of 1,25D. Furthermore, a subset of genes with evidence of interethnic differences in transcriptional response was also identified, suggesting that in addition to the well-established interethnic variation in circulating levels of vitamin D, the intensity of transcriptional response to 1,25D and LPS also varies between ethnic groups. We propose that dysregulation of the pathways identified in this study could contribute to immune-mediated disease risk. Tuesday, May 3 2016 08:37:05 AM Gene Regulatory Evolution During Speciation in a Songbird Davidson, J. H., Balakrishnan, C. N. Over the last decade, tremendous progress has been made toward a comparative understanding of gene regulatory evolution. However, we know little about how gene regulation evolves in birds, and how divergent genomes interact in their hybrids. Because of the unique features of birds – female heterogamety, a highly conserved karyotype, and the slow evolution of reproductive incompatibilities – an understanding of regulatory evolution in birds is critical to a comprehensive understanding of regulatory evolution and its implications for speciation. Using a novel complement of analyses of replicated RNA-seq libraries, we demonstrate abundant divergence in brain gene expression between zebra finch (Taeniopygia guttata) subspecies. By comparing parental populations and their F1 hybrids, we also show that gene misexpression is relatively rare among brain-expressed transcripts in male birds. If this pattern is consistent across tissues and sexes, it may partially explain the slow buildup of postzygotic reproductive isolation observed in birds relative to other taxa. Although we expected that the action of genetic drift on the island-dwelling zebra finch subspecies would be manifested in a higher rate of trans regulatory divergence, we found that most divergence was in cis regulation, following a pattern commonly observed in other taxa. Thus, our study highlights both unique and shared features of avian regulatory evolution. Tuesday, May 3 2016 08:37:05 AM The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos Min, B., Cho, S., Park, J. S., Jeon, K., Kang, Y.-K. Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT) embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts. Tuesday, May 3 2016 08:37:05 AM Preparing for Winter: The Transcriptomic Response Associated with Different Day Lengths in Drosophila montana Parker, D. J., Ritchie, M. G., Kankare, M. At northern latitudes, the most robust cue for assessing the onset of winter is the shortening of day lengths. Many species use day length as a cue to increase their cold tolerance and/or enter into diapause, but little is known about changes in gene expression that occur under different day lengths. We investigate the gene expression changes associated with differences in light/dark cycles in Drosophila montana, a northerly distributed species with a strong adult photoperiodic reproductive diapause. To examine gene expression changes induced by light both prior to and during diapause, we used both nondiapausing and diapausing flies. We found that the majority of genes that are differentially expressed between different day lengths in nondiapausing and diapausing flies differ. However, the biological processes involved were broadly similar. These included neuron development and metabolism, which are largely consistent with an increase in cold tolerance previously observed to occur in these flies. We also found that many genes associated with reproduction change in expression level between different day lengths, suggesting that D. montana use changes in day length to cue changes in reproduction both before and after entering into diapause. Finally, we also identified several interesting candidate genes for light-induced changes including Lsp2, para, and Ih. Tuesday, May 3 2016 08:37:05 AM The Arabidopsis Auxin Receptor F-Box Proteins AFB4 and AFB5 Are Required for Response to the Synthetic Auxin Picloram Prigge, M. J., Greenham, K., Zhang, Y., Santner, A., Castillejo, C., Mutka, A. M., OMalley, R. C., Ecker, J. R., Kunkel, B. N., Estelle, M. The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity. Tuesday, May 3 2016 08:37:05 AM Multiple Targets on the Gln3 Transcription Activator Are Cumulatively Required for Control of Its Cytoplasmic Sequestration Rai, R., Tate, J. J., Cooper, T. G. A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond, and the resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator, Gln3, responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e., nitrogen catabolite repression (NCR), long-term starvation, glutamine starvation, and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness, and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc13 sequestration in nitrogen excess. Here, we identify a serine/threonine-rich (Gln3477–493) region required for effective cytoplasmic Gln3-Myc13 sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on the responses of Gln3-Myc13 to rapamycin, methionine sulfoximine, or limiting nitrogen. However, cytoplasmic Gln3-Myc13 sequestration is additively, and almost completely, abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477–493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration. Tuesday, May 3 2016 08:37:05 AM Genetic Background, Maternal Age, and Interaction Effects Mediate Rates of Crossing Over in Drosophila melanogaster Females Hunter, C. M., Robinson, M. C., Aylor, D. L., Singh, N. D. Meiotic recombination is a genetic process that is critical for proper chromosome segregation in many organisms. Despite being fundamental for organismal fitness, rates of crossing over vary greatly between taxa. Both genetic and environmental factors contribute to phenotypic variation in crossover frequency, as do genotype–environment interactions. Here, we test the hypothesis that maternal age influences rates of crossing over in a genotypic-specific manner. Using classical genetic techniques, we estimated rates of crossing over for individual Drosophila melanogaster females from five strains over their lifetime from a single mating event. We find that both age and genetic background significantly contribute to observed variation in recombination frequency, as do genotype–age interactions. We further find differences in the effect of age on recombination frequency in the two genomic regions surveyed. Our results highlight the complexity of recombination rate variation and reveal a new role of genotype by maternal age interactions in mediating recombination rate. Tuesday, May 3 2016 08:37:05 AM A High-Density SNP Genetic Linkage Map and QTL Analysis of Growth-Related Traits in a Hybrid Family of Oysters (Crassostrea gigas x Crassostrea angulata) Using Genotyping-by-Sequencing Wang, J., Li, L., Zhang, G. Oysters are among the most important species in global aquaculture. Crassostrea gigas, and its subspecies C. angulata, are the major cultured species. To determine the genetic basis of growth-related traits in oysters, we constructed a second-generation linkage map from 3367 single-nucleotide polymorphisms (SNPs) based on genotyping-by-sequencing, genotyped from a C. gigas C. angulata hybrid family. These 3367 SNPs were distributed on 1695 markers, which were assigned to 10 linkage groups. The genetic linkage map had a total length of 1084.3 cM, with an average of 0.8 cM between markers; it thus represents the densest genetic map constructed for oysters to date. Twenty-seven quantitative trait loci (QTL) for five growth-related traits were detected. These QTL could explain 4.2–7.7% (mean = 5.4%) of the phenotypic variation. In total, 50.8% of phenotypic variance for shell width, 7.7% for mass weight, and 34.1% for soft tissue weight were explained. The detected QTL were distributed among eight linkage groups, and more than half (16) were concentrated within narrow regions in their respective linkage groups. Thirty-eight annotated genes were identified within the QTL regions, two of which are key genes for carbohydrate metabolism. Other genes were found to participate in assembly and regulation of the actin cytoskeleton, signal transduction, and regulation of cell differentiation and development. The newly developed high-density genetic map, and the QTL and candidate genes identified provide a valuable genetic resource and a basis for marker-assisted selection for C. gigas and C. angulata. Tuesday, May 3 2016 08:37:05 AM Quantitative Assessment of Eye Phenotypes for Functional Genetic Studies Using Drosophila melanogaster Iyer, J., Wang, Q., Le, T., Pizzo, L., Gronke, S., Ambegaokar, S. S., Imai, Y., Srivastava, A., Troisi, B. L., Mardon, G., Artero, R., Jackson, G. R., Isaacs, A. M., Partridge, L., Lu, B., Kumar, J. P., Girirajan, S. About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions. Tuesday, May 3 2016 08:37:05 AM The Sorghum Gene for Leaf Color Changes upon Wounding (P) Encodes a Flavanone 4-Reductase in the 3-Deoxyanthocyanidin Biosynthesis Pathway Kawahigashi, H., Kasuga, S., Sawada, Y., Yonemaru, J.-i., Ando, T., Kanamori, H., Wu, J., Mizuno, H., Momma, M., Fujimoto, Z., Hirai, M. Y., Matsumoto, T. Upon wounding or pathogen invasion, leaves of sorghum [Sorghum bicolor (L.) Moench] plants with the P gene turn purple, whereas leaves with the recessive allele turn brown or tan. This purple phenotype is determined by the production of two 3-deoxyanthocyanidins, apigeninidin and luteolinidin, which are not produced by the tan-phenotype plants. Using map-based cloning in progeny from a cross between purple Nakei-MS3B (PP) and tan Greenleaf (pp) cultivars, we isolated this gene, which was located in a 27-kb genomic region around the 58.1 Mb position on chromosome 6. Four candidate genes identified in this region were similar to the maize leucoanthocyanidin reductase gene. None of them was expressed before wounding, and only the Sb06g029550 gene was induced in both cultivars after wounding. The Sb06g029550 protein was detected in Nakei-MS3B, but only slightly in Greenleaf, in which it may be unstable because of a Cys252Tyr substitution. A recombinant Sb06g029550 protein had a specific flavanone 4-reductase activity, and converted flavanones (naringenin or eriodictyol) to flavan-4-ols (apiforol or luteoforol) in vitro. Our data indicate that the Sb06g029550 gene is involved in the 3-deoxyanthocyanidin synthesis pathway. Tuesday, May 3 2016 08:37:05 AM Organ Length Control by an ADAMTS Extracellular Protease in Caenorhabditis elegans Shibata, Y., Kawakado, Y., Hori, N., Tanaka, K., Inoue, R., Takano, T., Kubota, Y., Nishiwaki, K. Tuesday, May 3 2016 08:37:06 AM Pollen Killer Gene S35 Function Requires Interaction with an Activator That Maps Close to S24, Another Pollen Killer Gene in Rice Kubo, T., Yoshimura, A., Kurata, N. Pollen killer genes disable noncarrier pollens, and are responsible for male sterility and segregation distortion in hybrid populations of distantly related plant species. The genetic networks and the molecular mechanisms underlying the pollen killer system remain largely unknown. Two pollen killer genes, S24 and S35, have been found in an intersubspecific cross of Oryza sativa ssp. indica and japonica. The effect of S24 is counteracted by an unlinked locus EFS. Additionally, S35 has been proposed to interact with S24 to induce pollen sterility. These genetic interactions are suggestive of a single S24-centric genetic pathway (EFS–S24–S35) for the pollen killer system. To examine this hypothetical genetic pathway, the S35 and the S24 regions were further characterized and genetically dissected in this study. Our results indicated that S35 causes pollen sterility independently of both the EFS and S24 genes, but is dependent on a novel gene close to the S24 locus, named incentive for killing pollen (INK). We confirmed the phenotypic effect of the INK gene separately from the S24 gene, and identified the INK locus within an interval of less than 0.6 Mb on rice chromosome 5. This study characterized the genetic effect of the two independent genetic pathways of INK–S35 and EFS–S24 in indica–japonica hybrid progeny. Our results provide clear evidence that hybrid male sterility in rice is caused by several pollen killer networks with multiple factors positively and negatively regulating pollen killer genes. Tuesday, May 3 2016 08:37:06 AM Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen Santiago, E., Akamine, P., Snider, J., Wong, V., Jessulat, M., Deineko, V., Gagarinova, A., Aoki, H., Minic, Z., Phanse, S., San Antonio, A., Cubano, L. A., Rymond, B. C., Babu, M., Stagljar, I., Rodriguez-Medina, J. R. Tuesday, May 3 2016 08:37:06 AM A Forward Genetic Screen for Molecules Involved in Pheromone-Induced Dauer Formation in Caenorhabditis elegans Neal, S. J., Park, J., DiTirro, D., Yoon, J., Shibuya, M., Choi, W., Schroeder, F. C., Butcher, R. A., Kim, K., Sengupta, P. Animals must constantly assess their surroundings and integrate sensory cues to make appropriate behavioral and developmental decisions. Pheromones produced by conspecific individuals provide critical information regarding environmental conditions. Ascaroside pheromone concentration and composition are instructive in the decision of Caenorhabditis elegans to either develop into a reproductive adult or enter into the stress-resistant alternate dauer developmental stage. Pheromones are sensed by a small set of sensory neurons, and integrated with additional environmental cues, to regulate neuroendocrine signaling and dauer formation. To identify molecules required for pheromone-induced dauer formation, we performed an unbiased forward genetic screen and identified phd (pheromone response-defective dauer) mutants. Here, we describe new roles in dauer formation for previously identified neuronal molecules such as the WD40 domain protein QUI-1 and MACO-1 Macoilin, report new roles for nociceptive neurons in modulating pheromone-induced dauer formation, and identify tau tubulin kinases as new genes involved in dauer formation. Thus, phd mutants define loci required for the detection, transmission, or integration of pheromone signals in the regulation of dauer formation. Tuesday, May 3 2016 08:37:06 AM Corrigendum