Contents

November 2015 • VOLUME 5 • ISSUE 11 • www.g3journal.org

INVESTIGATIONS

2219–2227 Genes with Restricted Introgression in a Field Cricket (Gryllus firmus/Gryllus pennsylvanicus) Hybrid Zone Are Concentrated on the X Chromosome and a Single Autosome Luana S. Maroja, Erica L. Larson, Steven M. Bogdanowicz, and Richard G. Harrison Characterizing the extent of genomic differentiation between recently diverged lineages provides an important context for understanding the early stages of speciation. The authors construct a genetic linkage map for the hybridizing field crickets, Gryllus firmus and Gryllus pennsylvanicus. They show that genes that remain distinct in the face of hybridization are concentrated on the sex chromosome and in one autosomal region. The authors discuss the implications of these results for understanding the role of sex chromosomes in the origin of new species and why recently diverged species exhibit heterogeneous genomic divergence.

2229–2239 Cytogenetic and Sequence Analyses of Mitochondrial DNA Insertions in Nuclear Chromosomes of Maize Ashley N. Lough, Kaitlyn M. Faries, Dal-Hoe Koo, Abid Hussain, Leah M. Roark, Tiffany L. Langewisch, Teresa Backes, Karl A. G. Kremling, Jiming Jiang, James A. Birchler, and Kathleen J. Newton The transfer of mitochondrial DNA (mtDNA) into nuclear genomes occurs regularly. The authors examined intraspecific variation of nuclear-mtDNA sequences (NUMTs) among diverse maize inbred lines using fluorescence in situ hybridization (FISH). A large NUMT on the long arm of chromosome 9 contains a majority of the mitochondrial genome. This mtDNA insertion accounts for approximately 1% of chromosome 9 in the B73 line. However, the published nuclear genome sequence for B73 includes only part of the mtDNA sequences detected by FISH. Thus, cytogenetic analyses should be used to complement sequence searches when examining large insertions of organellar DNA into nuclear chromosomes.

2241–2255 High-Throughput Cloning of Temperature-Sensitive Caenorhabditis elegans Mutants with Adult Syncytial Germline Membrane Architecture Defects Josh Lowry, John Yochem, Chien-Hui Chuang,KenjiSugioka,AmyA.Connolly,and Bruce Bowerman The mechanisms that control the development and maintenance of the complex membrane architecture of the syncytial C. elegans germline remain poorly understood. The authors have identified temperature-sensitive mutations in 19 essential genes that cause defects in germline membrane architecture. Using whole genome sequencing, the authors identified the causal mutations in 10 of these mutants. Four of the affected genes are conserved, with orthologs known to be involved in membrane biology, and two of these are required for maintenance of the membrane architecture during adult oogenesis. This work provides a starting point for further investigation of a widely conserved feature of animal gametogenesis.

Volume 5 | November 2015 | ii 2257–2266 Fine-Mapping the Wheat Snn1 Locus Conferring Sensitivity to the Parastagonospora nodorum Necrotrophic Effector SnTox1 Using an Eight Founder Multiparent Advanced Generation Inter-Cross Population James Cockram, Alice Scuderi, Toby Barber, Eiko Furuki, Keith A. Gardner, Nick Gosman, Radoslaw Kowalczyk, Huyen P. Phan, Gemma A. Rose, Kar-Chun Tan, Richard P. Oliver, and Ian J. Mackay Bread wheat is one of the world's major crops. Genetic resistance of wheat to disease is a critical factor in yield protection, and thus maintaining global food security. Disease resistance is typically a complex trait. However, the recent identification of toxic effectors secreted by fungal pathogens now allows the quantitative wheat responses to be Mendalized. Here, the authors use a multiparent advanced generation inter-cross (MAGIC) wheat population to map sensitivity loci to the effector SnTox1, produced by the necrotrophic fungus Parastagonorum nodorum. Tagging SNPs are converted to breeder-friendly markers, providing useful resources for marker-assisted breeding approaches.

2267–2274 Convergent Loss of Awn in Two Cultivated Rice Species Oryza sativa and Oryza glaberrima Is Caused by Mutations in Different Loci Tomoyuki Furuta, Norio Komeda, Kenji Asano, Kanako Uehara, Rico Gamuyao, Rosalyn B. Angeles-Shim, Keisuke Nagai, Kazuyuki Doi, Diane R. Wang, Hideshi Yasui, Atsushi Yoshimura, Jianzhong Wu, Susan R. McCouch, and Motoyuki Ashikari A long awn is one of the distinct morphological features of wild rice species. So far, it has not been reported how many genes are involved the formation and how genes are lost through rice domestication. The authors found three loci (RAE1, RAE2, and RAE3) that are involved in the formation of awn. The authors also showed the two cultivated rice species, Oryza sativa (Asian rice) and Oryza glaberrima (African rice), have taken different paths to become awnless. This is the first report of independent loci being selected for the same trait during crop domestication.

2275–2283 Evolutionary Fate of the Androgen Receptor2Signaling Pathway in Ray-Finned Fishes with a Special Focus on Cichlids Thibault Lorin, Walter Salzburger, and Astrid Böhne The authors investigate for the first time the genetic composition of the androgen receptor–signaling pathway in ray-finned fish genomes characterizing gene duplications and losses, with a special focus on paralogs derived from the teleost-specific whole genome duplication (TSGD) using the spotted gar as reference. One particular lineage, the cichlids, shows differing selection pressures acting on TSGD duplicates. In cichlids from East African Lake Tanganyika, the authors find that gene duplicates differ in their expression pattern on the tissue and species level. The authors explore in more detail indications of a neofunctionalization event for the rac1 GTPase in cichlids.

2285–2290 SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean Qijian Song, Gaofeng Jia, David L. Hyten, Jerry Jenkins, Eun-Young Hwang, Steven G. Schroeder, Juan M. Osorno, Jeremy Schmutz, Scott A. Jackson, Phillip E. McClean, and Perry B. Cregan These authors identified SNP DNA markers based on a diverse set of common bean cultivars via next generation sequencing technologies; developed Illumina Infinium BeadChip assays containing SNPs with high polymorphism within and between common bean market classes; created high density genetic linkage maps; anchored and oriented large common bean sequence scaffolds generated by the Joint Genome Institute, Department of Energy; and designed a BARCBean6K_3 BeadChip with a core set of 6,000 highly informative SNP markers. The BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.

2291–2298 Genomic Correlates of Virulence Attenuation in the Deadly Amphibian Chytrid Fungus, Batrachochytrium dendrobatidis Jeanine M. Refsnider, Thomas J. Poorten, Penny F. Langhammer, Patricia A. Burrowes, and Erica Bree Rosenblum Understanding the genetic basis of virulence shifts for emerging pathogens is critical. The fungus Batrachochytrium dendrobatidis (Bd) causes a lethal skin disease in amphibians and is responsible for amphibian declines around the world. The authors compared full genomes of two Bd samples. Both originated from the same source, but one decreased in virulence over 30 generations in the lab. Virulence attenuation was associated with loss of chromosomal segments and mutations in specific protease genes. These results demonstrate that genomic processes, like changes in chromosomal copy number, can occur over extremely short timescales and may contribute to virulence shifts in emerging pathogens.

iii | Contents 2299–2306 Persistent Activation of the Innate Immune Response in Adult Drosophila Following Radiation Exposure During Larval Development Lisa J. Sudmeier, Sai-Suma Samudrala, Steven P. Howard, and Barry Ganetzky Cranial radiation therapy is an effective treatment for pediatric central nervous system malignancies, but survivors often suffer from long-term neurological and neurocognitive side effects. Here the authors show that the innate immune response is activated in the adult Drosophila brain following radiation exposure during larval development and that this response is sustained in adult flies weeks after radiation exposure. The authors also present preliminary data suggesting that innate immunity is radioprotective during Drosophila development. Together, these data suggest that activation of the innate immune response may be beneficial initially following radiation exposure. However, chronic inflammation may eventually lead to impaired neuronal function and viability.

2307–2321 Codon and Amino Acid Usage Are Shaped by Selection Across Divergent Model Organisms of the Pancrustacea Carrie A. Whittle and Cassandra G. Extavour Codon and amino acid usage have been correlated with expression level in some animals, and may be shaped by selection for efficient and accurate translation. Research on codon usage, and particularly amino acid frequency, remains focused on a few traditional animal models, but insights can be gained by expanding studies to a broader range of organisms. Here the authors use transcriptomes of three emerging model arthropods to reveal that highly expressed genes favor specific synonymous codons, moderate-cost amino acids, shorter proteins, and are preferentially involved in cell cycling and protein synthesis. These findings suggest translational selection operates across divergent Pancrustacea.

2323–2327 bz-rates: A Web Tool to Estimate Mutation Rates from Fluctuation Analysis Alexandre Gillet-Markowska, Guillaume Louvel, and Gilles Fischer bz-rates is a web tool to calculate mutation rates that provides three useful advances over existing web tools. First, it allows taking into account the differential growth rate between mutant and wild-type cells, in the estimation of the mean number of mutations (m). Secondly, it allows the user to take into account a deviation from the Luria-Delbrück distribution, namely the plating efficiency, in the estimation of m. Finally, the website provides a graphical visualization of the goodness-of-fit between the experimental data and the model.

2329–2339 The Genetic Architecture of Hearing Impairment in Mice: Evidence for Frequency-Specific Genetic Determinants Amanda L. Crow, Jeffrey Ohmen, Juemei Wang, Joel Lavinsky, Jaana Hartiala, Qingzhong Li, Xin Li, Pezhman Salehide, Eleazar Eskin, Calvin Pan, Aldons J. Lusis, Hooman Allayee, and Rick A. Friedman This article describes the first genome-wide association analysis of strain variation in auditory thresholds utilizing correction for population structure. The authors identify several novel QTLs and outline candidates in the region using bioinformatics analyses and in situ hybridization. The authors demonstrate a frequency-specific genetics to hearing loss in inbred mouse strains.

2341–2355 Ultra-High Density, Transcript-Based Genetic Maps of Pepper Define Recombination in the Genome and Synteny Among Related Species Theresa Hill, Hamid Ashrafi, Sebastian Reyes Chin-Wo, Kevin Stoffel, Maria-Jose Truco, Alexander Kozik, Richard Michelmore, and Allen Van Deynze The authors constructed ultra-high density EST-based genetic maps for Capsicum by interrogation of 30,000 unigenes segregating in two pepper RIL populations. Two high quality, comprehensive Capsicum genetic maps (a 16,000 unigene interspecific and a 5600 unigene intraspecific map) resulted in a total of 17,382 pepper unigenes mapped. Comparisons with the pepper genome showed extensive pericentromeric chromosomal regions with suppressed recombination that appear to be more pronounced in pepper than other plant species, except for tomato. This resource has already proven to be a valuable asset for molecular breeding through high resolution QTL mapping and gene candidate identification.

Volume 5 | November 2015 | iv 2357–2364 Genetic Linkage Mapping of Economically Important Traits in Cultivated Tetraploid Potato (Solanum tuberosum L.) Alicia N. Massa, Norma C. Manrique-Carpintero, Joseph J. Coombs, Daniel G. Zarka, Anne E. Boone, William W. Kirk, Christine A. Hackett, Glenn J. Bryan, and David S. Douches The cultivated autotetraploid potato presents unique challenges for linkage analysis and QTL mapping. Using a genome-wide set of SNP markers and a novel approach that incorporates allele dosage information, the authors identified a QTL and a candidate SNP associated with blight resistance in potato. The location of this SNP, within an R-gene hotspot on chromosome 9, provides strong evidence for the NB-LRR gene-based resistance. The study also identified a QTL and a candidate SNP associated with plant maturity on chromosome 5. These findings will enable the use of marker-assisted selection for resistance independent of the selection for maturity.

2365–2373 Exact Inference for Hardy-Weinberg Proportions with Missing Genotypes: Single and Multiple Imputation Jan Graffelman, S. Nelson, S. M. Gogarten, and B. S. Weir GWAS investigators test for Hardy-Weinberg equilibrium as part of their quality control process. Potentially misleading results can be avoided by imputing those data that result for SNPs with missing genotypes. This may avoid having to discard SNPs that are otherwise well-type.

2375–2382 Post-transcriptional Mechanisms Contribute Little to Phenotypic Variation in Snake Venoms Darin R. Rokyta, Mark J. Margres, and Kate Calvin The production of proteins is a fundamental process in the generation of organismal phenotypes, and protein quantities are determined by levels of mRNA as well as processes affecting how mRNA is translated into proteins. Recent work suggested a prominent role for the latter post-transcriptional processes in the emergence of phenotypes. However, these authors found that snake-venom phenotypes were determined directly through transcriptional patterns across 10 species from three families by showing extremely high correlations between mRNA and protein abundances. These results describe a simplified genotype-phenotype relationship and indicate that post-transcriptional mechanisms do not play a role in venoms.

2383–2390 LinkImpute: Fast and Accurate Genotype Imputation for Nonmodel Organisms Daniel Money, Kyle Gardner, Zoë Migicovsky, Heidi Schwaninger, Gan-Yuan Zhong, and Sean Myles Next-generation DNA sequencing often produces large amounts of missing genotype data in a population of samples. Imputation is a method used to fill in the missing data using patterns from the nonmissing genotypes. Here the authors introduce new software—LinkImpute—that performs imputation on genome-wide single nucleotide polymorphism (SNP) data from a population of samples. Unlike most imputation methods, LinkImpute does not rely on ordered markers and is therefore ideally suited for nonmodel organisms with limited genomic resources.

2391–2403 Genome-Wide Association Study of Ureide Concentration in Diverse Maturity Group IV Soybean [Glycine max (L.) Merr.] Accessions Jeffery D. Ray, Arun Prabhu Dhanapal, Shardendu K. Singh, Valerio Hoyos-Villegas, James R. Smith, Larry C. Purcell, C. Andy King, Debbie Boykin, Perry B. Cregan, Qijian Song, and Felix B. Fritschi Ureides are the N-rich products of N-fixation that are transported from soybean nodules to the shoot. Ureides are known to accumulate in leaves in response to water-deficit stress, and this has been used to identify genotypes with reduced N-fixation sensitivity to drought. The authors have shown variation among a large number of soybean genotypes in ureide concentration and identified chromosomal regions associated with that variation. Some regions corresponded to areas with known ureide-related genes, but others represent previously unreported regions associated with ureide concentration.

2405–2419 The Nature, Extent, and Consequences of Genetic Variation in the opa Repeats of Notch in Drosophila Clinton Rice, Danielle Beekman, Liping Liu, and Albert Erives These authors report finding functionally variant Drosophila Notch alleles differing in the opa repeats, which encode polyglutamine tracts. The exact nucleotide sequences of these opa repeats have been difficult to discern from recent whole genome assemblies. Rare short and long variant Notch opa alleles cause defects in embryonic survival and sensory organ specification, and can produce notched wings. Interestingly, radiation-resistant fly lines are more likely to carry opa variants than radiation-sensitive lines, suggesting chronic up-regulation of the unfolded protein response. These results suggest that the Notch opa repeat configuration is an important species-specific gene regulatory network variable.

v | Contents 2421–2427 Flux Control in a Defense Pathway in Arabidopsis thaliana Is Robust to Environmental Perturbations and Controls Variation in Adaptive Traits Carrie F. Olson-Manning, Christopher F. Strock, and Thomas Mitchell-Olds In the cell, metabolic pathways perform many vital functions. Understanding how genetic and environmental changes influence the output from a metabolic pathway and how natural selection acts on this variation is an important challenge. In this study, the authors found that the control over pathway output lies with a single step in the pathway, regardless of environmental perturbation. They also found that an insect herbivore alters its feeding behavior in response to changes in the defense chemicals. These results indicate that, despite the complexity of environments in nature, natural selection may repeatedly target the same gene.

2429–2439 Slit Binding via the Ig1 Domain Is Essential for Midline Repulsion by Drosophila Robo1 but Dispensable for Receptor Expression, Localization, and Regulation in Vivo Haley E. Brown, Marie C. Reichert, and Timothy A. Evans How are functional connections formed between the two mirror-image sides of bilateral nervous systems? Neuronal axons must properly navigate the plane of symmetry, responding to ligands produced by midline cells to decide whetherornottocrossthemidline.Usingthe versatile genetic toolkit of Drosophila, the authors take a novel approach to studying one well- known axon guidance pathway, the midline repellant Slit and its Robo1 receptor. By modifying Robo1 so that it can no longer recognize Slit, the authors tease apart Slit-dependent and Slit- independent aspects of Robo1's expression, regulation, and function in the developing nervous systems of intact animals.

2441–2452 De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes Sheila A. Kitchen, Camerron M. Crowder, Angela Z. Poole, Virginia M. Weis, and Eli Meyer This article details the assembly, annotation, and application of four anthozoan (phylum Cnidaria) transcriptomes, a group with limited genomic resources. Utilizing a variety of methods to estimate completeness of gene content, the authors confirm that these transcriptomes represent high quality gene catalogs. Using these resources and other available Cnidaria datasets, they identify orthologous sequences that they then use for phylogenetic analysis and demonstrate the superiority of this method over commonly used mitochondrial loci. This work highlights a promising path forward for creating a diverse repertoire of cnidarian genomic resources and resolving major uncertainties in coral phylogeny.

2453–2461 Whole-Genome Sequencing Suggests Schizophrenia Risk Mechanisms in Humans with 22q11.2 Deletion Syndrome Daniele Merico, Mehdi Zarrei, Gregory Costain, Lucas Ogura, Babak Alipanahi, Matthew J. Gazzellone, Nancy J. Butcher, Bhooma Thiruvahindrapuram, Thomas Nalpathamkalam, Eva W. C. Chow, Danielle M. Andrade, Brendan J. Frey, Christian R. Marshall, Stephen W. Scherer, and Anne S. Bassett Chromosome 22q11.2 microdeletions impart a high but incomplete risk for schizophrenia. In a proof- of-principle study to assess this model, the authors used high quality whole-genome sequencing of nine individuals with 22q11.2 deletions and extreme phenotypes (schizophrenia, or no psychotic disorder at age .50 years). The schizophrenia group had a greater burden of genome-wide rare variants impacting protein-coding neurofunctional genes, especially when restricted to those affected by a DGCR8/miRNA mechanism, highly conserved long intergenic non-coding RNA genes, and schizophrenia polygenic risk. The findings support exploiting this approach in larger cohorts to help delineate the genetic architecture of schizophrenia.

2463–2473 Linkage Mapping Reveals Strong Chiasma Interference in Sockeye Salmon: Implications for Interpreting Genomic Data Morten T. Limborg, Ryan K. Waples, Fred W. Allendorf, and James E. Seeb Genetic variation is the fuel that drives evolution and, in sexually reproducing species, it is continuously created through the process of meiotic recombination. Information about how recombination works is crucial when using genetic data to understand the demographic history of a species and how populations adapt to their local environment. Here the authors present a detailed description of recombination patterns in a duplicated salmonid genome and demonstrate how similar insights can be easily obtained in mapping studies of any nonmodel species. The results have implications for improving interpretation of genomic data, including identification of genes that control adaptively important traits.

Volume 5 | November 2015 | vi 2475–2486 Long-Lasting Gene Conversion Shapes the Convergent Evolution of the Critical Methanogenesis Genes Sishuo Wang, Youhua Chen, Qinhong Cao, and Huiqiang Lou Can you imagine two paralogous genes undergoing concerted evolution for more than 2410 million years? Here the authors report an extremely long-lasting gene conversion mechanism in constraining the divergence of the duplicated methyltransferase complex mtrA genes in some methanogens. Strikingly, gene conversion is strictly limited in the nontransmembrane (MtrA) domains. MtrA-2 obtained a transmembrane domain distinct from MtrA-1 through domain shuffling and gene fusion. Furthermore, the methyltransferase genes essential for methanogenesis may be traced back to an ancient non-methanogenic origin. These findings provide clues to understand the ill-defined methanogenesis process, which is crucial in Earth's global carbon cycle.

2487–2495 RNAseq Analysis Highlights Specific Transcriptome Signatures of Yeast and Mycelial Growth Phases in the Dutch Elm Disease Fungus Ophiostoma novo-ulmi Martha Nigg, Jérôme Laroche, Christian R. Landry, and Louis Bernier Because our understanding of dimorphism in ascomycete fungi is mostly limited to human pathogens, these authors propose the Dutch elm disease pathogen Ophiostoma novo-ulmi as a new model. They investigated the transcriptomes of yeast and mycelial growth phases of O. novo-ulmi and compared it with gene expression data from Histoplasma capsulatum and Candida albicans to identify conserved signatures among these ascomycetes. Their results provide the first genome-wide analysis of both growth phases in O. novo-ulmi. They identify few key molecular functions that may be conserved across species but show that gene expression signatures are poorly conserved with classical models of human pathogens.

2497–2511 Proteomic Analysis of Dhh1 Complexes Reveals a Role for Hsp40 Chaperone Ydj1 in Yeast P-Body Assembly Gregory A. Cary, Dani B. N. Vinh, Patrick May, Rolf Kuestner, and Aimée M. Dudley While the mRNA decay and translational repression machineries are known components of p-bodies, it remains unclear why and how cells assemble them into large cytoplasmic RNA-protein granules under stress. Here the authors affinity-isolate the p-body component Dhh1 and characterized the proteins and RNAs associated with it under conditions that induce p-body formation. They find that proteins containing low-complexity regions are enriched in the purifications, and that one, the Hsp40 chaperone Ydj1, is specifically required for p-body formation. They also see a substantial enrichment of the mRNA encoding the p-body protein Pat1 and several catalytic RNAs.

2513–2522 Genomic Instability of the Sex-Determining Locus in Atlantic Salmon (Salmo salar) Krzysztof P. Lubieniecki, Song Lin, Emily I. Cabana, Jieying Li, Yvonne Y. Y. Lai, and William S. Davidson Phenotypic sex (SEX) maps to at least three different chromosomes in Tasmanian Atlantic salmon, and a similar observation has been made in Arctic charr. It thus appeared that the sex-determining gene in salmonids can move around the genome, giving the impression that there are multiple SEX loci both within and between salmonid species. The authors characterized sdY (the sex-determining gene in rainbow trout) genomic region in Atlantic salmon, and showed that this gene is located in chromosomes to which SEX maps in Tasmanian salmon. They also identified an sdY cassette and suggest a mechanism whereby it can jump around the genome.

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