Donate Today!   Join GSA      

Home | Contact GSA      

 
 
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

View the Editorial Board  |  Read G3  |  Submit your manuscript  |  Contact us

 

 


 

What's Inside the Current Issue of G3

 

 

Tuesday, August 9 2016 08:40:48 AM

Genetic Architecture of Resistance to Stripe Rust in a Global Winter Wheat Germplasm Collection

Bulli, P., Zhang, J., Chao, S., Chen, X., Pumphrey, M.


Virulence shifts in populations of Puccinia striiformis f. sp. tritici (Pst), the causal pathogen of wheat stripe rust, are a major challenge to resistance breeding. The majority of known resistance genes are already ineffective against current races of Pst, necessitating the identification and introgression of new sources of resistance. Germplasm core collections that reflect the range of genetic and phenotypic diversity of crop species are ideal platforms for examining the genetic architecture of complex traits such as resistance to stripe rust. We report the results of genetic characterization and genome-wide association analysis (GWAS) for resistance to stripe rust in a core subset of 1175 accessions in the National Small Grains Collection (NSGC) winter wheat germplasm collection, based on genotyping with the wheat 9K single nucleotide polymorphism (SNP) iSelect assay and phenotyping of seedling and adult plants under natural disease epidemics in four environments. High correlations among the field data translated into high heritability values within and across locations. Population structure was evident when accessions were grouped by stripe rust reaction. GWAS identified 127 resistance loci that were effective across at least two environments, including 20 with significant genome-wide adjusted P-values. Based on relative map positions of previously reported genes and QTL, five of the QTL with significant genome-wide adjusted P-values in this study represent potentially new loci. This study provides an overview of the diversity of Pst resistance in the NSGC winter wheat germplasm core collection, which can be exploited for diversification of stripe rust resistance in breeding programs.



Tuesday, August 9 2016 08:40:48 AM

Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant

Gupta, S., Radhakrishnan, A., Nitin, R., Raharja-Liu, P., Lin, G., Steinmetz, L. M., Gagneur, J., Sinha, H.


Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae. During mitosis, the common TAO3 allele interacts with CBK1—a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2—a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait.



Tuesday, August 9 2016 08:40:48 AM

Detecting Recombination Hotspots from Patterns of Linkage Disequilibrium

Wall, J. D., Stevison, L. S.


With recent advances in DNA sequencing technologies, it has become increasingly easy to use whole-genome sequencing of unrelated individuals to assay patterns of linkage disequilibrium (LD) across the genome. One type of analysis that is commonly performed is to estimate local recombination rates and identify recombination hotspots from patterns of LD. One method for detecting recombination hotspots, LDhot, has been used in a handful of species to further our understanding of the basic biology of recombination. For the most part, the effectiveness of this method (e.g., power and false positive rate) is unknown. In this study, we run extensive simulations to compare the effectiveness of three different implementations of LDhot. We find large differences in the power and false positive rates of these different approaches, as well as a strong sensitivity to the window size used (with smaller window sizes leading to more accurate estimation of hotspot locations). We also compared our LDhot simulation results with comparable simulation results obtained from a Bayesian maximum-likelihood approach for identifying hotspots. Surprisingly, we found that the latter computationally intensive approach had substantially lower power over the parameter values considered in our simulations.



Tuesday, August 9 2016 08:40:48 AM

Accumulation of Deleterious Mutations Near Sexually Antagonistic Genes

Connallon, T., Jordan, C. Y.


Mutation generates a steady supply of genetic variation that, while occasionally useful for adaptation, is more often deleterious for fitness. Recent research has emphasized that the fitness effects of mutations often differ between the sexes, leading to important evolutionary consequences for the maintenance of genetic variation and long-term population viability. Some forms of sex-specific selection—i.e., stronger purifying selection in males than females—can help purge a population’s load of female-harming mutations and promote population growth. Other scenarios—e.g., sexually antagonistic selection, in which mutations that harm females are beneficial for males—inflate genetic loads and potentially dampen population viability. Evolutionary processes of sexual antagonism and purifying selection are likely to impact the evolutionary dynamics of different loci within a genome, yet theory has mostly ignored the potential for interactions between such loci to jointly shape the evolutionary genetic basis of female and male fitness variation. Here, we show that sexually antagonistic selection at a locus tends to elevate the frequencies of deleterious alleles at tightly linked loci that evolve under purifying selection. Moreover, haplotypes that segregate for different sexually antagonistic alleles accumulate different types of deleterious mutations. Haplotypes that carry female-benefit sexually antagonistic alleles preferentially accumulate mutations that are primarily male harming, whereas male-benefit haplotypes accumulate mutations that are primarily female harming. The theory predicts that sexually antagonistic selection should shape the genomic organization of genetic variation that differentially impacts female and male fitness, and contribute to sexual dimorphism in the genetic basis of fitness variation.



Tuesday, August 9 2016 08:40:48 AM

Diversifying Selection Between Pure-Breed and Free-Breeding Dogs Inferred from Genome-Wide SNP Analysis

Pilot, M., Malewski, T., Moura, A. E., Grzybowski, T., Olełski, K., Kamiłski, S., Fadel, F. R., Alagaili, A. N., Mohammed, O. B., Bogdanowicz, W.


Domesticated species are often composed of distinct populations differing in the character and strength of artificial and natural selection pressures, providing a valuable model to study adaptation. In contrast to pure-breed dogs that constitute artificially maintained inbred lines, free-ranging dogs are typically free-breeding, i.e., unrestrained in mate choice. Many traits in free-breeding dogs (FBDs) may be under similar natural and sexual selection conditions to wild canids, while relaxation of sexual selection is expected in pure-breed dogs. We used a Bayesian approach with strict false-positive control criteria to identify FST-outlier SNPs between FBDs and either European or East Asian breeds, based on 167,989 autosomal SNPs. By identifying outlier SNPs located within coding genes, we found four candidate genes under diversifying selection shared by these two comparisons. Three of them are associated with the Hedgehog (HH) signaling pathway regulating vertebrate morphogenesis. A comparison between FBDs and East Asian breeds also revealed diversifying selection on the BBS6 gene, which was earlier shown to cause snout shortening and dental crowding via disrupted HH signaling. Our results suggest that relaxation of natural and sexual selection in pure-breed dogs as opposed to FBDs could have led to mild changes in regulation of the HH signaling pathway. HH inhibits adhesion and the migration of neural crest cells from the neural tube, and minor deficits of these cells during embryonic development have been proposed as the underlying cause of "domestication syndrome." This suggests that the process of breed formation involved the same genetic and developmental pathways as the process of domestication.



Tuesday, August 9 2016 08:40:48 AM

Long Terminal Repeat Retrotransposon Content in Eight Diploid Sunflower Species Inferred from Next-Generation Sequence Data

Tetreault, H. M., Ungerer, M. C.


The most abundant transposable elements (TEs) in plant genomes are Class I long terminal repeat (LTR) retrotransposons represented by superfamilies gypsy and copia. Amplification of these superfamilies directly impacts genome structure and contributes to differential patterns of genome size evolution among plant lineages. Utilizing short-read Illumina data and sequence information from a panel of Helianthus annuus (sunflower) full-length gypsy and copia elements, we explore the contribution of these sequences to genome size variation among eight diploid Helianthus species and an outgroup taxon, Phoebanthus tenuifolius. We also explore transcriptional dynamics of these elements in both leaf and bud tissue via RT-PCR. We demonstrate that most LTR retrotransposon sublineages (i.e., families) display patterns of similar genomic abundance across species. A small number of LTR retrotransposon sublineages exhibit lineage-specific amplification, particularly in the genomes of species with larger estimated nuclear DNA content. RT-PCR assays reveal that some LTR retrotransposon sublineages are transcriptionally active across all species and tissue types, whereas others display species-specific and tissue-specific expression. The species with the largest estimated genome size, H. agrestis, has experienced amplification of LTR retrotransposon sublineages, some of which have proliferated independently in other lineages in the Helianthus phylogeny.



Tuesday, August 9 2016 08:40:48 AM

Characterization of a Single Genomic Locus Encoding the Clustered Protocadherin Receptor Diversity in Xenopus tropicalis

Etlioglu, H. E., Sun, W., Huang, Z., Chen, W., Schmucker, D.


Clustered protocadherins (cPcdhs) constitute the largest subgroup of the cadherin superfamily, and in mammals are grouped into clusters of α-, β-, and -types. Tens of tandemly arranged paralogous Pcdh genes of the Pcdh clusters generate a substantial diversity of receptor isoforms. cPcdhs are known to have important roles in neuronal development, and genetic alterations of cPcdhs have been found to be associated with several neurological diseases. Here, we present a first characterization of cPcdhs in Xenopus tropicalis. We determined and annotated all cPcdh isoforms, revealing that they are present in a single chromosomal locus. We validated a total of 96 isoforms, which we show are organized in three distinct clusters. The X. tropicalis cPcdh locus is composed of one α- and two distinct -Pcdh clusters (pcdh-1 and pcdh-2). Bioinformatics analyses assisted by genomic BAC clone sequencing showed that the X. tropicalis α- and -Pcdhs are conserved at the cluster level, but, unlike mammals, X. tropicalis does not contain a β-Pcdh cluster. In contrast, the number of -Pcdh isoforms has expanded, possibly due to lineage-specific gene duplications. Interestingly, the number of X. tropicalis α-Pcdhs is identical between X. tropicalis and mouse. Moreover, we find highly conserved as well as novel promoter elements potentially involved in regulating the cluster-specific expression of cPcdh isoforms. This study provides important information for the understanding of the evolutionary history of cPcdh genes and future mechanistic studies. It provides an annotated X. tropicalis cPcdh genomic map and a first molecular characterization essential for functional and comparative studies.



Tuesday, August 9 2016 08:40:48 AM

Genetic Regulation of Transcriptional Variation in Natural Arabidopsis thaliana Accessions

Zan, Y., Shen, X., Forsberg, S. K. G., Carlborg, O.


An increased knowledge of the genetic regulation of expression in Arabidopsis thaliana is likely to provide important insights about the basis of the plant’s extensive phenotypic variation. Here, we reanalyzed two publicly available datasets with genome-wide data on genetic and transcript variation in large collections of natural A. thaliana accessions. Transcripts from more than half of all genes were detected in the leaves of all accessions, and from nearly all annotated genes in at least one accession. Thousands of genes had high transcript levels in some accessions, but no transcripts at all in others, and this pattern was correlated with the genome-wide genotype. In total, 2669 eQTL were mapped in the largest population, and 717 of them were replicated in the other population. A total of 646 cis-eQTL-regulated genes that lacked detectable transcripts in some accessions was found, and for 159 of these we identified one, or several, common structural variants in the populations that were shown to be likely contributors to the lack of detectable RNA transcripts for these genes. This study thus provides new insights into the overall genetic regulation of global gene expression diversity in the leaf of natural A. thaliana accessions. Further, it also shows that strong cis-acting polymorphisms, many of which are likely to be structural variations, make important contributions to the transcriptional variation in the worldwide A. thaliana population.



Tuesday, August 9 2016 08:40:48 AM

Prospects of Genomic Prediction in the USDA Soybean Germplasm Collection: Historical Data Creates Robust Models for Enhancing Selection of Accessions

Jarquin, D., Specht, J., Lorenz, A.


The identification and mobilization of useful genetic variation from germplasm banks for use in breeding programs is critical for future genetic gain and protection against crop pests. Plummeting costs of next-generation sequencing and genotyping is revolutionizing the way in which researchers and breeders interface with plant germplasm collections. An example of this is the high density genotyping of the entire USDA Soybean Germplasm Collection. We assessed the usefulness of 50K single nucleotide polymorphism data collected on 18,480 domesticated soybean (Glycine max) accessions and vast historical phenotypic data for developing genomic prediction models for protein, oil, and yield. Resulting genomic prediction models explained an appreciable amount of the variation in accession performance in independent validation trials, with correlations between predicted and observed reaching up to 0.92 for oil and protein and 0.79 for yield. The optimization of training set design was explored using a series of cross-validation schemes. It was found that the target population and environment need to be well represented in the training set. Second, genomic prediction training sets appear to be robust to the presence of data from diverse geographical locations and genetic clusters. This finding, however, depends on the influence of shattering and lodging, and may be specific to soybean with its presence of maturity groups. The distribution of 7608 nonphenotyped accessions was examined through the application of genomic prediction models. The distribution of predictions of phenotyped accessions was representative of the distribution of predictions for nonphenotyped accessions, with no nonphenotyped accessions being predicted to fall far outside the range of predictions of phenotyped accessions.



Tuesday, August 9 2016 08:40:48 AM

A Genome-Wide Association Study for Regulators of Micronucleus Formation in Mice

McIntyre, R. E., Nicod, J., Robles-Espinoza, C. D., Maciejowski, J., Cai, N., Hill, J., Verstraten, R., Iyer, V., Rust, A. G., Balmus, G., Mott, R., Flint, J., Adams, D. J.


In mammals the regulation of genomic instability plays a key role in tumor suppression and also controls genome plasticity, which is important for recombination during the processes of immunity and meiosis. Most studies to identify regulators of genomic instability have been performed in cells in culture or in systems that report on gross rearrangements of the genome, yet subtle differences in the level of genomic instability can contribute to whole organism phenotypes such as tumor predisposition. Here we performed a genome-wide association study in a population of 1379 outbred Crl:CFW(SW)-US_P08 mice to dissect the genetic landscape of micronucleus formation, a biomarker of chromosomal breaks, whole chromosome loss, and extranuclear DNA. Variation in micronucleus levels is a complex trait with a genome-wide heritability of 53.1%. We identify seven loci influencing micronucleus formation (false discovery rate <5%), and define candidate genes at each locus. Intriguingly at several loci we find evidence for sexual dimorphism in micronucleus formation, with a locus on chromosome 11 being specific to males.



Tuesday, August 9 2016 08:40:48 AM

flyDIVaS: A Comparative Genomics Resource for Drosophila Divergence and Selection

Stanley, C. E., Kulathinal, R. J.


With arguably the best finished and expertly annotated genome assembly, Drosophila melanogaster is a formidable genetics model to study all aspects of biology. Nearly a decade ago, the 12 Drosophila genomes project expanded D. melanogaster’s breadth as a comparative model through the community-development of an unprecedented genus- and genome-wide comparative resource. However, since its inception, these datasets for evolutionary inference and biological discovery have become increasingly outdated, outmoded, and inaccessible. Here, we provide an updated and upgradable comparative genomics resource of Drosophila divergence and selection, flyDIVaS, based on the latest genomic assemblies, curated FlyBase annotations, and recent OrthoDB orthology calls. flyDIVaS is an online database containing D. melanogaster-centric orthologous gene sets, CDS and protein alignments, divergence statistics (% gaps, dN, dS, dN/dS), and codon-based tests of positive Darwinian selection. Out of 13,920 protein-coding D. melanogaster genes, ~80% have one aligned ortholog in the closely related species, D. simulans, and ~50% have 1–1 12-way alignments in the original 12 sequenced species that span over 80 million yr of divergence. Genes and their orthologs can be chosen from four different taxonomic datasets differing in phylogenetic depth and coverage density, and visualized via interactive alignments and phylogenetic trees. Users can also batch download entire comparative datasets. A functional survey finds conserved mitotic and neural genes, highly diverged immune and reproduction-related genes, more conspicuous signals of divergence across tissue-specific genes, and an enrichment of positive selection among highly diverged genes. flyDIVaS will be regularly updated and can be freely accessed at www.flydivas.info. We encourage researchers to regularly use this resource as a tool for biological inference and discovery, and in their classrooms to help train the next generation of biologists to creatively use such genomic big data resources in an integrative manner.



Tuesday, August 9 2016 08:40:48 AM

The Use of Targeted Marker Subsets to Account for Population Structure and Relatedness in Genome-Wide Association Studies of Maize (Zea mays L.)

Chen, A. H., Lipka, A. E.


A typical plant genome-wide association study (GWAS) uses a mixed linear model (MLM) that includes a trait as the response variable, a marker as an explanatory variable, and fixed and random effect covariates accounting for population structure and relatedness. Although effective in controlling for false positive signals, this model typically fails to detect signals that are correlated with population structure or are located in high linkage disequilibrium (LD) genomic regions. This result likely arises from each tested marker being used to estimate population structure and relatedness. Previous work has demonstrated that it is possible to increase the power of the MLM by estimating relatedness (i.e., kinship) with markers that are not located on the chromosome where the tested marker resides. To quantify the amount of additional significant signals one can expect using this so-called K_chr model, we reanalyzed Mendelian, polygenic, and complex traits in two maize (Zea mays L.) diversity panels that have been previously assessed using the traditional MLM. We demonstrated that the K_chr model could find more significant associations, especially in high LD regions. This finding is underscored by our identification of novel genomic signals proximal to the tocochromanol biosynthetic pathway gene ZmVTE1 that are associated with a ratio of tocotrienols. We conclude that the K_chr model can detect more intricate sources of allelic variation underlying agronomically important traits, and should therefore become more widely used for GWAS. To facilitate the implementation of the K_chr model, we provide code written in the R programming language.



Tuesday, August 9 2016 08:40:48 AM

Septate Junction Proteins Play Essential Roles in Morphogenesis Throughout Embryonic Development in Drosophila

Hall, S., Ward, R. E.


The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood–brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster. Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction.



Tuesday, August 9 2016 08:40:48 AM

A Population of Deletion Mutants and an Integrated Mapping and Exome-seq Pipeline for Gene Discovery in Maize

Jia, S., Li, A., Morton, K., Avoles-Kianian, P., Kianian, S. F., Zhang, C., Holding, D.


To better understand maize endosperm filling and maturation, we used -irradiation of the B73 maize reference line to generate mutants with opaque endosperm and reduced kernel fill phenotypes, and created a population of 1788 lines including 39 Mo17 x F2s showing stable, segregating, and viable kernel phenotypes. For molecular characterization of the mutants, we developed a novel functional genomics platform that combined bulked segregant RNA and exome sequencing (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. To exemplify the utility of the mutants and provide proof-of-concept for the bioinformatics platform, we present detailed characterization of line 937, an opaque mutant harboring a 6203 bp in-frame deletion covering six exons within the Opaque-1 gene. In addition, we describe mutant line 146 which contains a 4.8 kb intragene deletion within the Sugary-1 gene and line 916 in which an 8.6 kb deletion knocks out a Cyclin A2 gene. The publically available algorithm developed in this work improves the identification of causative deletions and its corresponding gaps within mapping peaks. This study demonstrates the utility of -irradiation for forward genetics in large nondense genomes such as maize since deletions often affect single genes. Furthermore, we show how this classical mutagenesis method becomes applicable for functional genomics when combined with state-of-the-art genomics tools.



Tuesday, August 9 2016 08:40:48 AM

A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis

Misra, M., Edmund, H., Ennis, D., Schlueter, M. A., Marot, J. E., Tambasco, J., Barlow, I., Sigurbjornsdottir, S., Mathew, R., Valles, A. M., Wojciech, W., Roth, S., Davis, I., Leptin, M., Gavis, E. R.


Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating the asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types, and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system, to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins, and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling.



Tuesday, August 9 2016 08:40:48 AM

A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans

Li, S., Xu, S., Ma, Y., Wu, S., Feng, Y., Cui, Q., Chen, L., Zhou, S., Kong, Y., Zhang, X., Yu, J., Wu, M., Zhang, S. O.


To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 μm in diameter. From a saturated forward genetic screen of 6.7 x 105 mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 μm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion.



Tuesday, August 9 2016 08:40:48 AM

Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation

Zhu, Y. O., Sherlock, G., Petrov, D. A.


Budding yeast has undergone several independent transitions from commercial to clinical lifestyles. The frequency of such transitions suggests that clinical yeast strains are derived from environmentally available yeast populations, including commercial sources. However, despite their important role in adaptive evolution, the prevalence of polyploidy and aneuploidy has not been extensively analyzed in clinical strains. In this study, we have looked for patterns governing the transition to clinical invasion in the largest screen of clinical yeast isolates to date. In particular, we have focused on the hypothesis that ploidy changes have influenced adaptive processes. We sequenced 144 yeast strains, 132 of which are clinical isolates. We found pervasive large-scale genomic variation in both overall ploidy (34% of strains identified as 3n/4n) and individual chromosomal copy numbers (36% of strains identified as aneuploid). We also found evidence for the highly dynamic nature of yeast genomes, with 35 strains showing partial chromosomal copy number changes and eight strains showing multiple independent chromosomal events. Intriguingly, a lineage identified to be baker’s/commercial derived with a unique damaging mutation in NDC80 was particularly prone to polyploidy, with 83% of its members being triploid or tetraploid. Polyploidy was in turn associated with a >2x increase in aneuploidy rates as compared to other lineages. This dataset provides a rich source of information on the genomics of clinical yeast strains and highlights the potential importance of large-scale genomic copy variation in yeast adaptation.



Tuesday, August 9 2016 08:40:48 AM

Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development

Ma, J., Brennan, K. J., DAloia, M. R., Pascuzzi, P. E., Weake, V. M.


The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.



Tuesday, August 9 2016 08:40:48 AM

MeSH-Informed Enrichment Analysis and MeSH-Guided Semantic Similarity Among Functional Terms and Gene Products in Chicken

Morota, G., Beissinger, T. M., Penagaricano, F.


Biomedical vocabularies and ontologies aid in recapitulating biological knowledge. The annotation of gene products is mainly accelerated by Gene Ontology (GO), and more recently by Medical Subject Headings (MeSH). Here, we report a suite of MeSH packages for chicken in Bioconductor, and illustrate some features of different MeSH-based analyses, including MeSH-informed enrichment analysis and MeSH-guided semantic similarity among terms and gene products, using two lists of chicken genes available in public repositories. The two published datasets that were employed represent (i) differentially expressed genes, and (ii) candidate genes under selective sweep or epistatic selection. The comparison of MeSH with GO overrepresentation analyses suggested not only that MeSH supports the findings obtained from GO analysis, but also that MeSH is able to further enrich the representation of biological knowledge and often provide more interpretable results. Based on the hierarchical structures of MeSH and GO, we computed semantic similarities among vocabularies, as well as semantic similarities among selected genes. These yielded the similarity levels between significant functional terms, and the annotation of each gene yielded the measures of gene similarity. Our findings show the benefits of using MeSH as an alternative choice of annotation in order to draw biological inferences from a list of genes of interest. We argue that the use of MeSH in conjunction with GO will be instrumental in facilitating the understanding of the genetic basis of complex traits.



Tuesday, August 9 2016 08:40:48 AM

Neurons That Underlie Drosophila melanogaster Reproductive Behaviors: Detection of a Large Male-Bias in Gene Expression in fruitless-Expressing Neurons

Newell, N. R., New, F. N., Dalton, J. E., McIntyre, L. M., Arbeitman, M. N.


Male and female reproductive behaviors in Drosophila melanogaster are vastly different, but neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How this set of neurons can generate such different behaviors between the two sexes is an unresolved question. A particular challenge is that fru P1-expressing neurons comprise only 2–5% of the adult nervous system, and so studies of adult head tissue or whole brain may not reveal crucial differences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons, allowing a sensitive, cell-type-specific assay. We find four times more male-biased than female-biased genes in TRAP mRNAs from fru P1-expressing neurons. This suggests a potential mechanism to generate dimorphism in behavior. The male-biased genes may direct male behaviors by establishing cell fate in a similar context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise from a shared set of neurons.



Tuesday, August 9 2016 08:40:48 AM

A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function

Wissel, S., Kieser, A., Yasugi, T., Duchek, P., Roitinger, E., Gokcezade, J., Steinmann, V., Gaul, U., Mechtler, K., Forstemann, K., Knoblich, J. A., Neumuller, R. A.


Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila. miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.



Tuesday, August 9 2016 08:40:48 AM

Mutations in Dnaaf1 and Lrrc48 Cause Hydrocephalus, Laterality Defects, and Sinusitis in Mice

Ha, S., Lindsay, A. M., Timms, A. E., Beier, D. R.


We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48. Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.



Tuesday, August 9 2016 08:40:48 AM

Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae

Cui, P., Jin, H., Vutukuru, M. R., Kaplan, C. D.


The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt-) phenotype for the lys2-128 allele of the LYS2 gene. Mutants that suppress lys2-128 allow transcription from a normally inactive Ty1 promoter, conferring a LYS+ phenotype. The arrangement of transcription units at lys2-128 is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt- phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6. We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128 found in Spt- mutants. We determine that the lys2-128 Spt- phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the element as the position of Spt- transcription in tested Spt- mutants.



Tuesday, August 9 2016 08:40:48 AM

Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila

Kontur, C., Kumar, S., Lan, X., Pritchard, J. K., Turkewitz, A. P.


Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded to a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies, in part, on ancestral lysosomal sorting machinery, but is also likely to involve novel factors. In prior work, multiple strains with defects in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation—a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wild-type copy of MMA1, and disrupting MMA1 in an otherwise wild-type strain phenocopies UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation.



Tuesday, August 9 2016 08:40:48 AM

Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

Feng, Y., Chen, C., Han, Y., Chen, Z., Lu, X., Liang, F., Li, S., Qin, W., Lin, S.


The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5'-NGG-3' protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology.



Tuesday, August 9 2016 08:40:48 AM

Genetic Analysis of Kernel Traits in Maize-Teosinte Introgression Populations

Liu, Z., Garcia, A., McMullen, M. D., Flint-Garcia, S. A.


Seed traits have been targeted by human selection during the domestication of crop species as a way to increase the caloric and nutritional content of food during the transition from hunter-gather to early farming societies. The primary seed trait under selection was likely seed size/weight as it is most directly related to overall grain yield. Additional seed traits involved in seed shape may have also contributed to larger grain. Maize (Zea mays ssp. mays) kernel weight has increased more than 10-fold in the 9000 years since domestication from its wild ancestor, teosinte (Z. mays ssp. parviglumis). In order to study how size and shape affect kernel weight, we analyzed kernel morphometric traits in a set of 10 maize-teosinte introgression populations using digital imaging software. We identified quantitative trait loci (QTL) for kernel area and length with moderate allelic effects that colocalize with kernel weight QTL. Several genomic regions with strong effects during maize domestication were detected, and a genetic framework for kernel traits was characterized by complex pleiotropic interactions. Our results both confirm prior reports of kernel domestication loci and identify previously uncharacterized QTL with a range of allelic effects, enabling future research into the genetic basis of these traits.



Tuesday, August 9 2016 08:40:48 AM

Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions

Olafsson, G., Thorpe, P. H.


The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2.



Tuesday, August 9 2016 08:40:48 AM

A Meiotic Drive Element in the Maize Pathogen Fusarium verticillioides Is Located Within a 102 kb Region of Chromosome V

Pyle, J., Patel, T., Merrill, B., Nsokoshi, C., McCall, M., Proctor, R. H., Brown, D. W., Hammond, T. M.


Fusarium verticillioides is an agriculturally important fungus because of its association with maize and its propensity to contaminate grain with toxic compounds. Some isolates of the fungus harbor a meiotic drive element known as Spore killer (SkK) that causes nearly all surviving meiotic progeny from an SkK x Spore killer-susceptible (SkS) cross to inherit the SkK allele. SkK has been mapped to chromosome V but the genetic element responsible for meiotic drive has yet to be identified. In this study, we used cleaved amplified polymorphic sequence markers to genotype individual progeny from an SkK x SkS mapping population. We also sequenced the genomes of three progeny from the mapping population to determine their single nucleotide polymorphisms. These techniques allowed us to refine the location of SkK to a contiguous 102 kb interval of chromosome V, herein referred to as the Sk region. Relative to SkS genotypes, SkK genotypes have one extra gene within this region for a total of 42 genes. The additional gene in SkK genotypes, herein named SKC1 for Spore Killer Candidate 1, is the most highly expressed gene from the Sk region during early stages of sexual development. The Sk region also has three hyper-variable regions, the longest of which includes SKC1. The possibility that SKC1, or another gene from the Sk region, is an essential component of meiotic drive and spore killing is discussed.



Tuesday, August 9 2016 08:40:48 AM

Using Sequence Variants in Linkage Disequilibrium with Causative Mutations to Improve Across-Breed Prediction in Dairy Cattle: A Simulation Study

van den Berg, I., Boichard, D., Guldbrandtsen, B., Lund, M. S.


Sequence data are expected to increase the reliability of genomic prediction by containing causative mutations directly, especially in cases where low linkage disequilibrium between markers and causative mutations limits prediction reliability, such as across-breed prediction in dairy cattle. In practice, the causative mutations are unknown, and prediction with only variants in perfect linkage disequilibrium with the causative mutations is not realistic, leading to a reduced reliability compared to knowing the causative variants. Our objective was to use sequence data to investigate the potential benefits of sequence data for the prediction of genomic relationships, and consequently reliability of genomic breeding values. We used sequence data from five dairy cattle breeds, and a larger number of imputed sequences for two of the five breeds. We focused on the influence of linkage disequilibrium between markers and causative mutations, and assumed that a fraction of the causative mutations was shared across breeds and had the same effect across breeds. By comparing the loss in reliability of different scenarios, varying the distance between markers and causative mutations, using either all genome wide markers from commercial SNP chips, or only the markers closest to the causative mutations, we demonstrate the importance of using only variants very close to the causative mutations, especially for across-breed prediction. Rare variants improved prediction only if they were very close to rare causative mutations, and all causative mutations were rare. Our results show that sequence data can potentially improve genomic prediction, but careful selection of markers is essential.



Tuesday, August 9 2016 08:40:48 AM

Variation and Evolution in the Glutamine-Rich Repeat Region of Drosophila Argonaute-2

Palmer, W. H., Obbard, D. J.


RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel.



Tuesday, August 9 2016 08:40:48 AM

Genomic and Transcriptomic Associations Identify a New Insecticide Resistance Phenotype for the Selective Sweep at the Cyp6g1 Locus of Drosophila melanogaster

Battlay, P., Schmidt, J. M., Fournier-Level, A., Robin, C.


Scans of the Drosophila melanogaster genome have identified organophosphate resistance loci among those with the most pronounced signature of positive selection. In this study, the molecular basis of resistance to the organophosphate insecticide azinphos-methyl was investigated using the Drosophila Genetic Reference Panel, and genome-wide association. Recently released full transcriptome data were used to extend the utility of the Drosophila Genetic Reference Panel resource beyond traditional genome-wide association studies to allow systems genetics analyses of phenotypes. We found that both genomic and transcriptomic associations independently identified Cyp6g1, a gene involved in resistance to DDT and neonicotinoid insecticides, as the top candidate for azinphos-methyl resistance. This was verified by transgenically overexpressing Cyp6g1 using natural regulatory elements from a resistant allele, resulting in a 6.5-fold increase in resistance. We also identified four novel candidate genes associated with azinphos-methyl resistance, all of which are involved in either regulation of fat storage, or nervous system development. In Cyp6g1, we find a demonstrable resistance locus, a verification that transcriptome data can be used to identify variants associated with insecticide resistance, and an overlap between peaks of a genome-wide association study, and a genome-wide selective sweep analysis.



Tuesday, August 9 2016 08:40:48 AM

Evolution of the Insertion-Deletion Mutation Rate Across the Tree of Life

Sung, W., Ackerman, M. S., Dillon, M. M., Platt, T. G., Fuqua, C., Cooper, V. S., Lynch, M.


Mutations are the ultimate source of variation used for evolutionary adaptation, while also being predominantly deleterious and a source of genetic disorders. Understanding the rate of insertion-deletion mutations (indels) is essential to understanding evolutionary processes, especially in coding regions, where such mutations can disrupt production of essential proteins. Using direct estimates of indel rates from 14 phylogenetically diverse eukaryotic and bacterial species, along with measures of standing variation in such species, we obtain results that imply an inverse relationship of mutation rate and effective population size. These results, which corroborate earlier observations on the base-substitution mutation rate, appear most compatible with the hypothesis that natural selection reduces mutation rates per effective genome to the point at which the power of random genetic drift (approximated by the inverse of effective population size) becomes overwhelming. Given the substantial differences in DNA metabolism pathways that give rise to these two types of mutations, this consistency of results raises the possibility that refinement of other molecular and cellular traits may be inversely related to species-specific levels of random genetic drift.



Tuesday, August 9 2016 08:40:48 AM

Crosses Heterozygous for Hybrid Neurospora Translocation Strains Show Transmission Ratio Distortion Disfavoring Homokaryotic Ascospores Made Following Alternate Segregation

Giri, D. A., Rekha, S., Kasbekar, D. P.


By introgressing Neurospora crassa translocations into N. tetrasperma, we constructed heterokaryons bearing haploid nuclei of opposite mating types, and either the translocation and normal sequence chromosomes (i.e., [T + N]) or a duplication and its complementary deficiency (i.e., [Dp + Df]). The [T + N] heterokaryons result from alternate segregation of homologous centromeres, whereas adjacent-1 segregation generates [Dp + Df]. Self-cross of either heterokaryon produces [T + N] and [Dp + Df] progeny. Occasionally during N. tetrasperma ascus development, a pair of smaller homokaryotic ascospores replaces a heterokaryotic ascospore. Crosses with the Eight-spore mutant increase such replacement, and can generate asci with eight homokaryotic ascospores, either 4T + 4N from alternate segregation, or 4Dp + 4Df from adjacent-1 segregation. Crosses of some of the introgressed translocation strains with normal sequence N. tetrasperma produced more Dp than T or N homokaryotic progeny. We suggest this is due to an insufficiency for a presumptive ascospore maturation factor, which increases the chance that, in asci with > 4 viable ascospores, none properly mature. Since only four viable ascospores (Dp or [Dp + Df]) share the limiting factor following adjacent-1 segregation, whereas four to eight ascospores compete for it following alternate segregation, this would explain why Dp homokaryons outnumber T and N types, whereas the heterokaryons are not as affected. We believe that this novel form of transmission ratio distortion is caused by a Bateson–Dobzhansky–Muller Incompatibility (BDMI) triggered by an N. crassa gene in the N. tetrasperma background. Heterokaryons tend not to out-cross, and crosses of Dp strains are barren, thus the BDMI impedes interspecies gene flow.



Tuesday, August 9 2016 08:40:48 AM

Chromosome-End Knockoff Strategy to Reshape Alkaloid Profiles of a Fungal Endophyte

Florea, S., Phillips, T. D., Panaccione, D. G., Farman, M. L., Schardl, C. L.


Molecular genetic techniques to precisely eliminate genes in asexual filamentous fungi require the introduction of a marker gene into the target genome. We developed a novel strategy to eliminate genes or gene clusters located in subterminal regions of chromosomes, and then eliminate the marker gene and vector backbone used in the transformation procedure. Because many toxin gene clusters are subterminal, this method is particularly suited to generating nontoxic fungal strains. We tested this technique on Epichloë coenophiala, a seed-transmissible symbiotic fungus (endophyte) of the important forage grass, tall fescue (Lolium arundinaceum). The endophyte is necessary for maximal productivity and sustainability of this grass but can produce ergot alkaloids such as ergovaline, which are toxic to livestock. The genome sequence of E. coenophiala strain e19 revealed two paralogous ergot alkaloid biosynthesis gene clusters, designated EAS1 and EAS2. EAS1 was apparently subterminal, and the lpsB copy in EAS2 had a frame-shift mutation. We designed a vector with a fungal-active hygromycin phosphotransferase gene (hph), an lpsA1 gene fragment for homologous recombination at the telomere-distal end of EAS1, and a telomere repeat array positioned to drive spontaneous loss of hph and other vector sequences, and to stabilize the new chromosome end. We transformed E. coenophiala with this vector, then selected "knockoff" endophyte strains, confirmed by genome sequencing to lack 162 kb of a chromosome end including most of EAS1, and also to lack vector sequences. These EAS1 knockoff strains produced no detectable ergovaline, whereas complementation with functional lpsB restored ergovaline production.



Tuesday, August 9 2016 08:40:48 AM

Assessing Predictive Properties of Genome-Wide Selection in Soybeans

Xavier, A., Muir, W. M., Rainey, K. M.


Many economically important traits in plant breeding have low heritability or are difficult to measure. For these traits, genomic selection has attractive features and may boost genetic gains. Our goal was to evaluate alternative scenarios to implement genomic selection for yield components in soybean (Glycine max L. merr). We used a nested association panel with cross validation to evaluate the impacts of training population size, genotyping density, and prediction model on the accuracy of genomic prediction. Our results indicate that training population size was the factor most relevant to improvement in genome-wide prediction, with greatest improvement observed in training sets up to 2000 individuals. We discuss assumptions that influence the choice of the prediction model. Although alternative models had minor impacts on prediction accuracy, the most robust prediction model was the combination of reproducing kernel Hilbert space regression and BayesB. Higher genotyping density marginally improved accuracy. Our study finds that breeding programs seeking efficient genomic selection in soybeans would best allocate resources by investing in a representative training set.



Tuesday, August 9 2016 08:40:48 AM

Comparative Analysis of Regions with Distorted Segregation in Three Diploid Populations of Potato

Manrique-Carpintero, N. C., Coombs, J. J., Veilleux, R. E., Buell, C. R., Douches, D. S.


Genes associated with gametic and zygotic selection could underlie segregation distortion, observed as alterations of expected Mendelian genotypic frequencies in mapping populations. We studied highly dense genetic maps based on single nucleotide polymorphisms to elucidate the genetic nature of distorted segregation in potato. Three intra- and interspecific diploid segregating populations were used. DRH and D84 are crosses between the sequenced doubled monoploid DM 1-3 516 R44 Solanum tuberosum Group Phureja and either RH89-039-16 S. tuberosum or 84SD22, a S. tuberosum x S. chacoense hybrid. MSX902 is an interspecific cross between 84SD22 and Ber83 S. berthaultii x 2 x species mosaic. At the 0.05 significance level, 21%, 57%, and 51% of the total markers mapped in DRH, D84, and MSX902 exhibited distorted segregation, respectively. Segregation distortion regions for DRH were located on chromosomes 9 and 12; for D84 on chromosomes 2, 3, 4, 6, 7, and 8; and on chromosomes 1, 2, 7, 9, and 12 for MSX902. In general, each population had unique segregation distortion regions and directions of distortion. Interspecific crosses showed greater levels of distorted segregation and lower recombination rates as determined from the male parents. The different genomic regions where the segregation distortion regions occurred in the three populations likely reflect unique genetic combinations producing distorted segregation.



Tuesday, August 9 2016 08:40:48 AM

A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis

Ables, E. T., Hwang, G. H., Finger, D. S., Hinnant, T. D., Drummond-Barbosa, D.


Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.



Tuesday, August 9 2016 08:40:48 AM

RNAi Screen Identifies Novel Regulators of RNP Granules in the Caenorhabditis elegans Germ Line

Wood, M. P., Hollis, A., Severance, A. L., Karrick, M. L., Schisa, J. A.


Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest.



Tuesday, August 9 2016 08:40:48 AM

Identification of Functional Domains in the Cohesin Loader Subunit Scc4 by a Random Insertion/Dominant Negative Screen

Shwartz, M., Matityahu, A., Onn, I.


Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader—a heterodimer composed of Scc4 and Scc2. However, very little is known about the loader’s mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2. Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism.



Tuesday, August 9 2016 08:40:48 AM

Phenotypes Associated with Second Chromosome P Element Insertions in Drosophila melanogaster

Kahsai, L., Millburn, G. H., Cook, K. R.


In Drosophila melanogaster, P element transposition has been a productive means of insertional mutagenesis. Thousands of genes have been tagged with natural and engineered P element constructs. Nevertheless, chromosomes carrying P element insertions tend to have high levels of background mutations from P elements inserting and excising during transposition. Consequently, the phenotypes seen when P element-bearing chromosomes are homozygous are often not attributable to the P insertions themselves. In this study, 178 strains in the Bloomington Drosophila Stock Center collection with P insertions on the second chromosome were complementation tested against molecularly defined chromosomal deletions and previously characterized single-gene mutations to determine if recessive lethality or sterility is associated with the P insertions rather than background mutations. This information should prove valuable to geneticists using these strains for experimental studies of gene function.



Tuesday, August 9 2016 08:40:48 AM

A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription

Sammons, M. A., Zhu, J., Berger, S. L.


The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription.