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Jullien M Flynn, Emily J Brown, Andrew G Clark
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab184

Abstract:
Simple sequence tandem repeats are among the most rapidly evolving compartments of the genome. Some repeat expansions are associated with mammalian disease or meiotic segregation distortion, yet the rates of copy number change across generations are not well known. Here, we use 14 distinct sub-lineages of the C57BL/6 and C57BL/10 inbred mouse strains, which have been evolving independently over about 300 generations, to estimate the rates of copy number changes in genome-wide tandem repeats. Rates of change varied across repeats and across lines. Notably, CAG, whose expansions in coding regions are associated with many neurological and genetic disorders, was highly stable in copy number, likely indicating stabilizing selection. Rates of change were positively correlated with copy number, but the direction and magnitude of changes varied across lines. Some mouse lines experienced consistent losses or gains across most simple repeats, but this did not correlate with copy number changes in complex repeats. Rates of copy number change were similar between simple repeats and the more abundant complex repeats after normalization by copy number. Finally, the Y-specific centromeric repeat had a 4-fold higher rate of change than the homologous centromeric repeat on other chromosomes. Structural differences in satellite complexity, or restriction to the Y chromosome and elevated mutation rates of the male germline, may explain the higher rate of change. Overall, our work underscores the mutational fluidity of long tandem arrays of repeats, and the correlations and constraints between genome-wide tandem repeats which suggest that turnover is not a completely neutral process.
, Tilman Schell, Markus Pfenninger
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab180

Abstract:
Among all molluscs, land snails are a scientifically and economically interesting group comprising edible species, alien species and agricultural pests. Yet, despite their high diversity, the number of genome drafts publicly available is still scarce. Here, we present the draft genome assembly of the land snail Candidula unifasciata, a widely distributed species along central Europe, belonging to the Geomitridae family, a highly diversified taxon in the Western-Palearctic region. We performed whole genome sequencing, assembly and annotation of an adult specimen based on PacBio and Oxford Nanopore long read sequences as well as Illumina data. A genome draft of about 1.29 Gb was generated with a N50 length of 246 kb. More than 60% of the assembled genome was identified as repetitive elements. In total, 22,464 protein-coding genes were identified in the genome, of which 62.27% were functionally annotated. This is the first assembled and annotated genome for a geometrid snail and will serve as reference for further evolutionary, genomic and population genetic studies of this important and interesting group.
, Lucila I Buzzi, Carlos P Modenutti, Ana Acosta-Montalvo, , María S Rossi
G3 Genes|Genomes|Genetics, Volume 11; doi:10.1093/g3journal/jkab093

Abstract:
In the article by D. A. Caraballo, L. I. Buzzi, C. P. Modenutti, A. Acosta-Montalvo, O. A. Castro, and M. S. Rossi (G3: Genes|Genomes|Genetics 10(2): 755-768) entitled “Origin and Evolution of Two Independently Duplicated Genes Encoding UDP- Glucose: Glycoprotein Glucosyltransferases in Caenorhabditis and Vertebrates”, on page 755 the original manuscript within the author list did not acknowledge Dr. Olga A. Castro as a co-corresponding author. There was also an indication that Drs. Olga A. Castro and María S. Rossi were equal contributors which has been removed. In addition, the contact for Dr. Caraballo originally listed as “[email protected]” has been updated to “[email protected]”.
, Lu Ma, Donglai Xiao, Xiaoyu Liu, Xiaoling Jiang, Zhenghe Ying, Yanquan Lin
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab173

Abstract:
Sparassis latifolia is a valuable edible mushroom cultivated in China. In 2018, our research group reported an incomplete and low-quality genome of S. latifolia obtained by Illumina HiSeq 2500 sequencing. These limitations in the available genome have constrained genetic and genomic studies in this mushroom resource. Herein, an updated draft genome sequence of S. latifolia was generated by Oxford Nanopore sequencing and the high-through chromosome conformation capture (Hi-C) technique. A total of 8.24 Gb of Oxford Nanopore long reads representing ∼198.08X coverage of the S. latifolia genome were generated. Subsequently, a high-quality genome of 41.41 Mb, with scaffold and contig N50 sizes of 3.31 and 1.51 Mb, respectively, was assembled. Hi-C scaffolding of the genome resulted in 12 pseudochromosomes containing 93.56% of the bases in the assembled genome. Genome annotation further revealed that 17.47% of the genome was composed of repetitive sequences. In addition, 13,103 protein-coding genes were predicted, among which 98.72% were functionally annotated. BUSCO assay results further revealed that there were 92.07% complete BUSCOs. The improved chromosome-scale assembly and genome features described here will aid further molecular elucidation of various traits, breeding of S. latifolia, and evolutionary studies with related taxa.
Anne Krogh Nøhr, Kristian Hanghøj, Genis Garcia Erill, Zilong Li, ,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab174

Abstract:
Estimation of relatedness between pairs of individuals is important in many genetic research areas. When estimating relatedness, it is important to account for admixture if this is present. However, the methods that can account for admixture are all based on genotype data as input, which is a problem for low-depth next-generation sequencing (NGS) data from which genotypes are called with high uncertainty. Here we present a software tool, NGSremix, for maximum likelihood estimation of relatedness between pairs of admixed individuals from low-depth NGS data, which takes the uncertainty of the genotypes into account via genotype likelihoods. Using both simulated and real NGS data for admixed individuals with an average depth of 4x or below we show that our method works well and clearly outperforms all the commonly used state-of-the-art relatedness estimation methods PLINK, KING, relateAdmix, and ngsRelate that all perform quite poorly. Hence, NGSremix is a useful new tool for estimating relatedness in admixed populations from low-depth NGS data. NGSremix is implemented in C/C ++ in a multi-threaded software and is freely available on Github https://github.com/KHanghoj/NGSremix.
Lauren J Frazee, , Dinusha C Maheepala, Alannie-Grace Grant, , , ,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab177

Abstract:
The evolutionary transition from outcross-fertilization to self-fertilization is one of the most common in angiosperms and is often associated with a parallel shift in floral morphological and developmental traits, such as reduced flower size and pollen to ovule ratios, known as the ‘selfing syndrome’. How these convergent phenotypes arise, the extent to which they are shaped by selection, and the nature of their underlying genetic basis are unsettled questions in evolutionary biology. The genus Collinsia (Plantaginaceae) includes seven independent transitions from outcrossing or mixed mating to high selfing rates accompanied by selfing syndrome traits. Accordingly, Collinsia represents an ideal system for investigating this parallelism, but requires genomic resource development. We present a high quality de novo genome assembly for the highly selfing species C. rattanii. To begin addressing the basis of selfing syndrome developmental shifts, we evaluate and contrast patterns of gene expression from floral transcriptomes across three stages of bud development for C. rattanii and its outcrossing sister species C. linearis. Relative to C. linearis, total gene expression is less variable among individuals and bud stages in C. rattanii. In addition, there is a common pattern among differentially expressed genes: lower expression levels that are more constant across bud development in C. rattanii relative to C. linearis. Transcriptional regulation of enzymes involved in pollen formation specifically in early bud development may influence floral traits that distinguish selfing and outcrossing Collinsia species through pleiotropic functions. Future work will include additional Collinsia outcrossing-selfing species pairs to identify genomic signatures of parallel evolution.
Weisheng Wu, Jennie L Lovett, Kerby Shedden, Beverly I Strassmann,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab176

Abstract:
Genomic imprinting is an epigenetic mechanism that results in allele specific expression (ASE) based on parent of origin. It is known to play a role in the prenatal and postnatal allocation of maternal resources in mammals. ASE detected by whole transcriptome RNA-seq (wht-RNAseq) has been widely used to analyze imprinted genes using reciprocal crosses in mice to generate large numbers of informative SNPs. Studies in humans are more challenging due to the paucity of SNPs and the poor preservation of RNA in term placentas and other tissues. Targeted RNA-seq (tar-RNAseq) can potentially mitigate these challenges by focusing sequencing resources on the regions of interest in the transcriptome. Here we compared tar-RNAseq and wht-RNAseq in a study of ASE in known imprinted genes in placental tissue collected from a healthy human cohort in Mali, West Africa. As expected, tar-RNAseq substantially improved the coverage of SNPs. Compared to wht-RNAseq, tar-RNAseq produced on average four times more SNPs in twice as many genes per sample and read depth at the SNPs increased 4-fold. In previous research on humans, discordant ASE values for SNPs of the same gene have limited the ability to accurately quantify ASE. We show that tar-RNAseq reduces this limitation as it unexpectedly increased the concordance of ASE between SNPs of the same gene, even in cases of degraded RNA. Studies aimed at discovering associations between individual variation in ASE and phenotypes in mammals and flowering plants will benefit from the improved power and accuracy of tar-RNAseq.
Celine Caseys, Gongjun Shi, Nicole Soltis, Raoni Gwinner, Jason Corwin, Susanna Atwell,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab175

Abstract:
Botrytis cinerea is a fungal pathogen that causes necrotic disease on more than a thousand known hosts widely spread across the plant kingdom. How B. cinerea interacts with such extensive host diversity remains largely unknown. To address this question, we generated an infectivity matrix of 98 strains of B. cinerea on 90 genotypes representing eight host plants. This experimental infectivity matrix revealed that the disease outcome is largely explained by variations in either the host resistance or pathogen virulence. However, the specific interactions between host and pathogen account for 16% of the disease outcome. Furthermore, the disease outcomes cluster among genotypes of a species but are independent of the relatedness between hosts. When analyzing the host specificity and virulence of B. cinerea, generalist strains are predominant. In this fungal necrotroph, specialization may happen by a loss in virulence on most hosts rather than an increase of virulence on a specific host. To uncover the genetic architecture of Botrytis host specificity and virulence, a genome-wide association study (GWAS) was performed and revealed up to 1492 genes of interest. The genetic architecture of these traits is widespread across B. cinerea genome. The complexity of the disease outcome might be explained by hundreds of functionally diverse genes putatively involved in adjusting the infection to diverse hosts.
, Andrew C Olson,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab167

Abstract:
Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here, we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to compliment the already well-developed genetic analysis of Gαo signaling.
Tiffany Ou, Gary Huang, Beth Wilson, Paul Gontarz, James B Skeath,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab172

Abstract:
The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developing Drosophila body wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendon cells. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, including back seat driver, kon-tiki, thisbe, and tumbleweed, arguing our screen had the depth and precision to uncover myogenic genes. We also identified new alleles of spalt-major, barren, and patched that presumably disrupt independent muscle morphogenesis pathways. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be mapped and characterized. Our screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that regulate skeletal muscle topology.
Aleksandra Beric, MaKenzie E Mabry, Alex E Harkess, Julia Brose, M Eric Schranz, Gavin C Conant, Patrick P Edger, Blake C Meyers,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab140

Abstract:
Genome sizes of plants have long piqued the interest of researchers due to the vast differences among organisms. However, the mechanisms that drive size differences have yet to be fully understood. Two important contributing factors to genome size are expansions of repetitive elements, such as transposable elements (TEs), and whole-genome duplications (WGD). Although studies have found correlations between genome size and both TE abundance and polyploidy, these studies typically test for these patterns within a genus or species. The plant order Brassicales provides an excellent system to further test if genome size evolution patterns are consistent across larger time scales, as there are numerous WGDs. This order is also home to one of the smallest plant genomes, Arabidopsis thaliana—chosen as the model plant system for this reason—as well as to species with very large genomes. With new methods that allow for TE characterization from low-coverage genome shotgun data and 71 taxa across the Brassicales, we confirm the correlation between genome size and TE content, however, we are unable to reconstruct phylogenetic relationships and do not detect any shift in TE abundance associated with WGD.
Saptaparni Bandyopadhyay, Joseph Douglass, Sebastian Kapell, Nazimuddin Khan, Fabiana Feitosa-Suntheimer, Jenny A Klein, Jasmine Temple, Jayce Brown-Culbertson, Alexander H Tavares, , et al.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab169

Abstract:
Knock-in of large transgenes by Cas9-mediated homology-directed repair (HDR) is an extremely inefficient process. Although the use of single-stranded oligonucleotides (ssODN) as an HDR donor has improved the integration of smaller transgenes, they do not support efficient insertion of large DNA sequences. In an effort to gain insights into the mechanism(s) governing the HDR-mediated integration of larger transgenes and to improve the technology, we conducted knock-in experiments targeting the human EMX1 locus and applied rigorous genomic PCR analyses in the human HEK293 cell line. This exercise revealed an unexpected molecular complication arising from the transgene HDR being initiated at the single homology arm and the subsequent genomic integration of plasmid backbone sequences. To pivot around this problem, we devised a novel PCR-constructed template containing blocked long 3' single-stranded overhangs (BL3SSO) that greatly improved the efficiency of bona fide Cas9-stimulated HDR at the EMX1 locus. We further refined BL3SSO technology and successfully used it to insert GFP transgenes into two important interferon-stimulated genes (ISGs) loci, Viperin/RSAD2, and ISG15. This study demonstrates the utility of the BL3SSO platform for inserting long DNA sequences into both constitutive and inducible endogenous loci to generate novel human cell lines for the study of important biological processes.
, Nhung Nguyen, Steven B Roberts
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab148

Abstract:
Symbiosis with protists is common among cnidarians such as corals and sea anemones and is associated with homeostatic and phenotypic changes in the host that could have epigenetic underpinnings, such as methylation of CpG dinucleotides. We leveraged the sensitivity to base modifications of nanopore sequencing to probe the effect of symbiosis with the chlorophyte Elliptochloris marina on methylation in the sea anemone Anthopleura elegantissima. We first validated the approach by comparison of nanopore-derived methylation levels with CpG depletion analysis of a published transcriptome, finding that high methylation levels are associated with CpG depletion as expected. Next, using reads generated exclusively from aposymbiotic anemones, a largely complete draft genome comprising 243 Mb was assembled. Reads from aposymbiotic and symbiotic sea anemones were then mapped to this genome and assessed for methylation using the program Nanopolish, which detects signal disruptions from base modifications as they pass through the nanopore. Based on assessment of 452,841 CpGs for which there was adequate read coverage (approximately 8% of the CpGs in the genome), symbiosis with E. marina was, surprisingly, associated with only subtle changes in the host methylome. However, we did identify one extended genomic region with consistently higher methylation among symbiotic individuals. The region was associated with a DNA polymerase zeta that is noted for its role in translesion synthesis, which opens interesting questions about the biology of this symbiosis. Our study highlights the power and relative simplicity of nanopore sequencing for studies of nucleic acid base modifications in non-model species.
Gaotian Zhang, Jake D Mostad,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab168

Abstract:
Life history traits underlie the fitness of organisms and are under strong natural selection. A new mutation that positively impacts a life history trait will likely increase in frequency and become fixed in a population (e.g., a selective sweep). The identification of the beneficial alleles that underlie selective sweeps provides insights into the mechanisms that occurred during the evolution of a species. In the global population of Caenorhabditis elegans, we previously identified selective sweeps that have drastically reduced chromosomal-scale genetic diversity in the species. Here, we measured the fecundity of 121 wild C. elegans strains, including many recently isolated divergent strains from the Hawaiian islands and found that strains with larger swept genomic regions have significantly higher fecundity than strains without evidence of the recent selective sweeps. We used genome-wide association (GWA) mapping to identify three quantitative trait loci (QTL) underlying the fecundity variation. In addition, we mapped previous fecundity data from wild C. elegans strains and C. elegans recombinant inbred advanced intercross lines that were grown in various conditions and detected eight QTL using GWA and linkage mappings. These QTL show the genetic complexity of fecundity across this species. Moreover, the haplotype structure in each GWA QTL region revealed correlations with recent selective sweeps in the C. elegans population. North American and European strains had significantly higher fecundity than most strains from Hawaii, a hypothesized origin of the C. elegans species, suggesting that beneficial alleles that caused increased fecundity could underlie the selective sweeps during the worldwide expansion of C. elegans.
Justin L Conover, ,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab170

Abstract:
With the rapid rise in availability of high-quality genomes for closely related species, methods for orthology inference that incorporate synteny are increasingly useful. Polyploidy perturbs the 1:1 expected frequencies of orthologs between two species, complicating the identification of orthologs. Here we present a method of ortholog inference, Ploidy-aware Syntenic Orthologous Networks Identified via Collinearity (pSONIC). We demonstrate the utility of pSONIC using four species in the cotton tribe (Gossypieae), including one allopolyploid, and place between 75% and 90% of genes from each species into nearly 32,000 orthologous groups, 97% of which consist of at most singletons or tandemly duplicated genes—58.8% more than comparable methods that do not incorporate synteny. We show that 99% of singleton gene groups follow the expected tree topology and that our ploidy-aware algorithm recovers 97.5% identical groups when compared to splitting the allopolyploid into its two respective subgenomes, treating each as separate “species.”
Mafalda M Dias, João Vidigal, Daniela P Sequeira, Paula M Alves, Ana P Teixeira,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab166

Abstract:
Insect Trichoplusia ni High Five™ (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this study, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e., iCherry, as an intracellular protein model) or two proteins (i.e., influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development.
Jianqin Jiao, Kanisha Kavdia, Vishwajeeth Pagala, Lance Palmer, David Finkelstein, Yiping Fan, Junmin Peng,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab165

Abstract:
Recent evidence indicates that the composition of the ribosome is heterogeneous and that multiple types of specialized ribosomes regulate the synthesis of specific protein subsets. In Drosophila, we find that expression of the ribosomal RpS28 protein variants RpS28a and RpS28-like preferentially occurs in the germline, a tissue resistant to aging, and that it significantly declines in skeletal muscle during aging. Muscle-specific overexpression of RpS28a at levels similar to those seen in the germline decreases early mortality and promotes the synthesis of a subset of proteins with known anti-aging roles, some of which have preferential expression in the germline. These findings indicate a contribution of specialized ribosomal proteins to the regulation of the muscle proteome during aging.
Elizabeth D Drewnik, Tobias Wiesenfahrt, Ryan B Smit, Ye-Jean Park, Linda M Pallotto,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab164

Abstract:
Actin and myosin mediate the epidermal cell contractions that elongate the Caenorhabditis elegans embryo from an ovoid to a tubular-shaped worm. Contraction occurs mainly in the lateral epidermal cells, while the dorsoventral epidermis plays a more passive role. Two parallel pathways trigger actinomyosin contraction, one mediated by LET-502/Rho kinase and the other by PAK-1/p21 activated kinase. A number of genes mediating morphogenesis have been shown to be sufficient when expressed either laterally or dorsoventrally. Additional genes show either lateral or dorsoventral phenotypes. This led us to a model where contractile genes have discrete functions in one or the other cell type. We tested this by examining several genes for either lateral or dorsoventral sufficiency. LET-502 expression in the lateral cells was sufficient to drive elongation. MEL-11/Myosin phosphatase, which antagonizes contraction, and PAK-1 were expected to function dorsoventrally, but we could not detect tissue-specific sufficiency. Double mutants of lethal alleles predicted to decrease lateral contraction with those thought to increase dorsoventral force were previously shown to be viable. We hypothesized that these mutant combinations shifted the contractile force from the lateral to the dorsoventral cells and so the embryos would elongate with less lateral cell contraction. This was tested by examining ten single and double mutant strains. In most cases, elongation proceeded without a noticeable alteration in lateral contraction. We suggest that many embryonic elongation genes likely act in both lateral and dorsoventral cells, even though they may have their primary focus in one or the other cell type.
Valérie Gautier, Emilie Levert, Tatiana Giraud,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab159

Abstract:
Melanins are pigments used by fungi to withstand various stresses and to strengthen vegetative and reproductive structures. In Sordariales fungi, their biosynthesis starts with a condensation step catalyzed by an evolutionary-conserved polyketide synthase. Here we show that complete inactivation of this enzyme in the model ascomycete Podospora anserina through targeted deletion of the PaPks1 gene results in reduced female fertility, in contrast to a previously analyzed nonsense mutation in the same gene that retains full fertility. We also show the utility of PaPks1 mutants for detecting rare genetic events in P. anserina, such as parasexuality and possible fertilization and/or apomixis of nuclei devoid of mating-type gene.
Binhui Guo, Yi Dai, Lin Chen, Zhenzhi Pan,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab162

Abstract:
In plants, nitrate acts not only as a signaling molecule that affects plant development but also as a nutrient. The development of plant roots, which directly absorb nutrients, is greatly affected by nitrate supply. Alternative gene splicing plays a crucial role in the plant stress response by increasing transcriptome diversity. The effects of nitrate supply on alternative splicing (AS), however, have not been investigated in soybean roots. We used high-quality high-throughput RNA-sequencing data to investigate genome-wide AS events in soybean roots in response to various levels of nitrate supply. In total, we identified 355 nitrate-responsive AS events between optimal and high nitrate levels (NH), 335 nitrate-responsive AS events between optimal and low nitrate levels (NL), and 588 nitrate-responsive AS events between low and high nitrate levels (NLH). RI and A3SS were the most common AS types; in particular, they accounted for 67% of all AS events under all conditions. This increased complex and diversity of AS events regulation might be associated with the soybean response to nitrate. Functional ontology enrichment analysis suggested that the differentially splicing genes were associated with several pathways, including spliceosome, base excision repair, mRNA surveillance pathway and so on. Finally, we validated several AS events using reverse transcription–polymerase chain reaction to confirm our RNA-seq results. In summary, we characterized the features and patterns of genome-wide AS in the soybean root exposed to different nitrate levels, and our results revealed that AS is an important mechanism of nitrate-response regulation in the soybean root.
, Alfred Ozimati, Peter Kulakow, Michael A Gore, Marnin D Wolfe, Ephraim Nuwamanya, Chiedozie Egesi, Robert S Kawuki
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab160

Abstract:
Global efforts are underway to develop cassava with enhanced levels of provitamin A carotenoids to sustainably meet increasing demands for food and nutrition where the crop is a major staple. Herein, we tested the effectiveness of genomic selection for rapid improvement of cassava for total carotenoids content and associated traits. We evaluated 632 clones from Uganda’s provitamin A cassava breeding pipeline and 648 West African introductions. At harvest, each clone was assessed for level of total carotenoids, dry matter content and resistance to cassava brown streak disease. All clones were genotyped with diversity array technology and imputed to a set of 23,431 single nucleotide polymorphic markers. We assessed predictive ability of four genomic prediction methods in scenarios of cross-validation, across population prediction and inclusion of quantitative trait loci markers. Cross-validations produced the highest mean prediction ability for total carotenoids content (0.52) and the lowest for cassava brown streak disease resistance (0.20), with G-BLUP outperforming other models tested. Across population predictions showed low ability of Ugandan population to predict the performance of West African clones, with the highest predictive ability recorded for total carotenoids content (0.34) and the lowest for cassava brown streak disease resistance (0.12) using G-BLUP. By incorporating chromosome 1 markers associated with carotenoids content as independent kernel in the G-BLUP model of a cross-validation scenario, prediction ability slightly improved from 0.52 to 0.58. These results reinforce ongoing efforts aimed at integrating genomic selection into cassava breeding and demonstrate the utility of this tool for rapid genetic improvement.
Vera Pavese, Emile Cavalet-Giorsa, , , , , , Roberto Botta
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab152

Abstract:
The European hazelnut (Corylus avellana L.; 2n = 2x = 22) is a worldwide economically important tree nut that is cross-pollinated due to sporophytic incompatibility. Therefore, any individual plant is highly heterozygous. Cultivars are clonally propagated using mound layering, rooted suckers, and micropropagation. In recent years, the interest in this crop has increased, due to a growing demand related to the recognized health benefits of nut consumption. C. avellana cv “Tonda Gentile delle Langhe” (“TGdL”) is well-known for its high kernel quality, and the premium price paid for this cultivar is an economic benefit for producers in northern Italy. Assembly of a high-quality genome is a difficult task in many plant species because of the high level of heterozygosity. We assembled a chromosome-level genome sequence of “TGdL” with a two-step approach. First, 10X Genomics Chromium Technology was used to create a high-quality sequence, which was then assembled into scaffolds with cv “Tombul” genome as the reference. Eleven pseudomolecules were obtained, corresponding to 11 chromosomes. A total of 11,046 scaffolds remained unplaced, representing 11% of the genome (46,504,161 bp). Gene prediction, performed with Maker-P software, identified 27,791 genes (AED ≤0.4 and 92% of BUSCO completeness), whose function was analyzed with BlastP and InterProScan software. To characterize “TGdL” specific genetic mechanisms, Orthofinder was used to detect orthologs between hazelnut and closely related species. The “TGdL” genome sequence is expected to be a powerful tool to understand hazelnut genetics and allow detection of markers/genes for important traits to be used in targeted breeding programs.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab161

Abstract:
The generation of Drosophila stable cell lines has become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here, we describe a toolkit and procedure that combines CRISPR/Cas9 and theaaa PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.
Aleen D Saxton,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab158

Abstract:
Amyotrophic lateral sclerosis (ALS) is a debilitating, fatal neurodegenerative disease that causes rapid muscle wasting. It shares a spectrum of symptoms and pathology with frontotemporal lobar degeneration (FTLD). These diseases are caused by aberrant activity of a set of proteins including TDP-43 and UBIQUILIN-2 (UBQLN2). UBQLN2 encodes a ubiquitin-like adaptor protein involved in the ubiquitin-proteasome protein degradation pathway. Mutations in the PXX domain of UBQLN2 cause familial ALS. UBQLN2 aggregates in skein-like inclusions with other ALS and FTLD associated proteins including TDP-43 and ubiquitin. To facilitate further investigation of UBQLN2-mediated mechanisms of neurodegeneration, we made Caenorhabditis elegans transgenic lines pan-neuronally expressing human UBQLN2 cDNAs carrying either the wild-type UBQLN2 sequence or UBQLN2 with ALS causing mutations. Transgenic animals exhibit motor dysfunction accompanied by neurodegeneration of GABAergic motor neurons. At low levels of UBQLN2 expression, wild-type UBQLN2 causes significant motor impairment and neurodegeneration that is exacerbated by ALS associated mutations in UBQLN2. At higher levels of UBQLN2 expression, both wild-type and ALS mutated versions of UBQLN2 cause severe impairment. Molecular genetic investigation revealed that UBQLN2 dependent locomotor defects do not require the involvement of the endogenous homolog of TDP-43 in C. elegans (tdp-1). However, co-expression of wild-type human TDP-43 exacerbates UBQLN2 deficits. This model of UBQLN2-mediated neurodegeneration may be useful for further mechanistic investigation into the molecular cascades driving neurodegeneration in ALS and ALS-FTLD.
, Victoria Offord, Gemma Turner, Agnes Swiatkowska, Anneliese O Speak, David J Adams
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab157

Abstract:
Metastasis is the spread of cancer cells to a secondary site within the body, and is the leading cause of death for cancer patients. The lung is a common site of metastasis for many cancer types, including melanoma. Identifying the genes involved in aiding metastasis of melanoma cells to the lungs is critical for the development of better treatments. As the accessibility of cell surface proteins makes them attractive therapeutic targets, we performed a CRISPR activation screen using a library of guide RNAs (gRNAs) targeting the transcription start sites of 2195 membrane protein-encoding genes, to identify genes whose upregulated expression aided pulmonary metastasis. Immunodeficient mice were subcutaneously injected in the flank with murine B16-F0 melanoma cells expressing dCas9 and the membrane protein library gRNAs, and their lungs collected after 14–21 days. Analysis was performed to identify the gRNAs that were enriched in the lungs relative to those present in the cells at the time of administration (day 0). We identified six genes whose increased expression promotes lung metastasis. These genes included several with well-characterized pro-metastatic roles (Fut7, Mgat5, and Pcdh7) that have not previously been linked to melanoma progression, genes linked to tumor progression but that have not previously been described as involved in metastasis (Olfr322 and Olfr441), as well as novel genes (Tmem116). Thus, we have identified genes that, when upregulated in melanoma cells, can aid successful metastasis and colonization of the lung, and therefore may represent novel therapeutic targets to inhibit pulmonary metastasis.
Héloïse Muller, David Ogereau, Jean-Luc Da Lage, Claire Capdevielle, Nicolas Pollet, Taiadjana Fortuna, Rémi Jeannette, Laure Kaiser,
G3 Genes|Genomes|Genetics, Volume 11; doi:10.1093/g3journal/jkab155

Abstract:
The Mediterranean corn borer (Sesamia nonagrioides, Noctuidae, Lepidoptera) is a major pest of maize in Europe and Africa. Here, we report an assembly of the nuclear and mitochondrial genome of a pool of inbred males and females third-instar larvae, based on short- and long-read sequencing. The complete mitochondrial genome is 15,330 bp and contains all expected 13 and 24 protein-coding and RNA genes, respectively. The nuclear assembly is 1021 Mb, composed of 2553 scaffolds and it has an N50 of 1105 kb. It is more than twice larger than that of all Noctuidae species sequenced to date, mainly due to a higher repeat content. A total of 17,230 protein-coding genes were predicted, including 15,776 with InterPro domains. We provide detailed annotation of genes involved in sex determination (doublesex, insulin-like growth factor 2 mRNA-binding protein, and P-element somatic inhibitor) and of alpha-amylase genes possibly involved in interaction with parasitoid wasps. We found no evidence of recent horizontal transfer of bracovirus genes from parasitoid wasps. These genome assemblies provide a solid molecular basis to study insect genome evolution and to further develop biocontrol strategies against S. nonagrioides.
Keisuke Tomita, Yoko Yashiroda, , Jeff S Piotrowski, Sheena C Li, Reika Okamoto, Mami Yoshimura, Hiromi Kimura, Yumi Kawamura, Makoto Kawamukai, et al.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab156

Abstract:
Momilactone B is a natural product with dual biological activities, including antimicrobial and allelopathic properties, and plays a major role in plant chemical defense against competitive plants and pathogens. The pharmacological effects of momilactone B on mammalian cells have also been reported. However, little is known about the molecular and cellular mechanisms underlying its broad bioactivity. In this study, the genetic determinants of momilactone B sensitivity in yeast were explored to gain insight into its mode of action. We screened fission yeast mutants resistant to momilactone B from a pooled culture containing genome-wide gene-overexpressing strains in a drug-hypersensitive genetic background. Overexpression of pmd1, bfr1, pap1, arp9, or SPAC9E9.06c conferred resistance to momilactone B. In addition, a drug-hypersensitive, barcoded deletion library was newly constructed and the genes that imparted altered sensitivity to momilactone B upon deletion were identified. Gene Ontology and fission yeast phenotype ontology enrichment analyses predicted the biological pathways related to the mode of action of momilactone B. The validation of predictions revealed that momilactone B induced abnormal phenotypes such as multiseptated cells and disrupted organization of the microtubule structure. This is the first investigation of the mechanism underlying the antifungal activity of momilactone B against yeast. The results and datasets obtained in this study narrow the possible targets of momilactone B and facilitate further studies regarding its mode of action.
, Dean Podlich,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab153

Abstract:
Commercial hybrid breeding operations can be described as decentralized networks of smaller, more or less isolated breeding programs. There is further a tendency for the disproportionate use of successful inbred lines for generating the next generation of recombinants, which has led to a series of significant bottlenecks, particularly in the history of the North American and European maize germplasm. Both the decentralization and the disproportionate contribution of inbred lines reduce effective population size and constrain the accessible genetic space. Under these conditions, long-term response to selection is not expected to be optimal under the classical infinitesimal model of quantitative genetics. In this study, we therefore aim to propose a rationale for the success of large breeding operations in the context of genetic complexity arising from the structure and properties of interactive genetic networks. For this, we use simulations based on the NK model of genetic architecture. We indeed found that constraining genetic space through program decentralization and disproportionate contribution of parental inbred lines, is required to expose additive genetic variation and thus facilitate heritable genetic gains under high levels of genetic complexity. These results introduce new insights into why the historically grown structure of hybrid breeding programs was successful in improving the yield potential of hybrid crops over the last century. We also hope that a renewed appreciation for “why things worked” in the past can guide the adoption of novel technologies and the design of future breeding strategies for navigating biological complexity.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab151

Abstract:
The spontaneous mutation rate is a very variable trait that is subject to drift, selection and is sometimes highly plastic. Consequently, its variation between close species, or even between populations from the same species, can be very large. Here, I estimated the spontaneous mutation rate of Drosophila pseudoobscura and Drosophila persimilis crosses to explore the mutation rate variation within the Drosophila genus. All mutation rate estimations in Drosophila varied fourfold, probably explained by the sensitivity of the mutation rate to environmental and experimental conditions. Moreover, I found a very high mutation rate in the hybrid cross between D. pseudoobscura and D. persimilis, in agreement with known elevated mutation rate in hybrids. This mutation rate increase can be explained by heterozygosity and fitness decrease effects in hybrids.
, Zachary Brenton, James Heuser, Stephen Kresovich, Nadia Shakoor, Todd Mockler, Elizabeth A Cooper
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab154

Abstract:
Genomic structural mutations, especially deletions, are an important source of variation in many species and can play key roles in phenotypic diversification and evolution. Previous work in many plant species has identified multiple instances of structural variations (SVs) occurring in or near genes related to stress response and disease resistance, suggesting a possible role for SVs in local adaptation. Sorghum (Sorghum bicolor (L.) Moench) is one of the most widely grown cereal crops in the world. It has been adapted to an array of different climates as well as bred for multiple purposes, resulting in a striking phenotypic diversity. In this study, we identified genome-wide SVs in the Biomass Association Panel, a collection of 347 diverse sorghum genotypes collected from multiple countries and continents. Using Illumina-based, short-read whole genome resequencing data from every genotype, we found a total of 24,648 SVs, including 22,359 deletions. The global site frequency spectrum of deletions and other types of SVs fit a model of neutral evolution, suggesting that the majority of these mutations were not under any types of selection. Clustering results based on single nucleotide polymorphisms separated the genotypes into eight clusters which largely corresponded with geographic origins, with many of the large deletions we uncovered being unique to a single cluster. Even though most deletions appeared to be neutral, a handful of cluster-specific deletions were found in genes related to biotic and abiotic stress responses, supporting the possibility that at least some of these deletions contribute to local adaptation in sorghum.
, Aisha Ellahi, , Jagdish Suresh Patel, ,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab163

Abstract:
The gene encoding the ubiquitous DNA repair protein, Ku70p, has undergone extensive copy number expansion during primate evolution. Gene duplications of KU70 have the hallmark of long interspersed element-1 mediated retrotransposition with evidence of target-site duplications, the poly-A tails, and the absence of introns. Evolutionary analysis of this expanded family of KU70-derived “NUKU” retrocopies reveals that these genes are both ancient and also actively being created in extant primate species. NUKU retrocopies show evidence of functional divergence away from KU70, as evinced by their altered pattern of tissue expression and possible tissue-specific translation. Molecular modeling predicted that amino acid changes in Nuku2p at the interaction interface with Ku80p would prevent the assembly of the Ku heterodimer. The lack of Nuku2p-Ku80p interaction was confirmed by yeast two-hybrid assay, which contrasts the robust interaction of Ku70p-Ku80p. While several NUKU retrocopies appear to have been degraded by mutation, NUKU2 shows evidence of positive natural selection, suggesting that this retrocopy is undergoing neofunctionalization. Although Nuku proteins do not appear to antagonize retrovirus transduction in cell culture, the observed expansion and rapid evolution of NUKUs could be being driven by alternative selective pressures related to infectious disease or an undefined role in primate physiology.
Biao Xuan, Jongbin Park, SukJung Choi, Inhwan You, Bo-Hye Nam, Eun Soo Noh, Eun Mi Kim, Mi-Young Song, Younhee Shin, Ji-Hyeon Jeon
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab147

Abstract:
Pond smelt (Hypomesus nipponensis) is a cold-freshwater fish species as a winter economic resource of aquaculture in South Korea. Due to its high susceptibility to abnormal water temperature from global warming, a large number of smelt die in hot summer. Here, we present the first draft genome of H. nipponensis and transcriptomic changes in molecular mechanisms or intracellular responses under heat stress. We combined Illumina and PacBio sequencing technologies to generate the draft genome of H. nipponensis. Based on the reference genome, we conducted transcriptome analysis of liver and muscle tissues under normal (NT, 5 °C) versus warm (HT, 23 °C) conditions, to identify heat stress-induced genes and gene categories. We observed a total of 1,987 contigs, with N50 of 0.46 Mbp with a largest contig (3.03 Mbp) in the assembled genome. A total number of 20,644 protein coding genes were predicted, and 19,224 genes were functionally annotated: 15,955 genes for Gene Ontology (GO) terms; and 11,560 genes for KEGG Orthology (KO). We conducted the lost and gained genes analysis compared with three species that human, zebrafish and salmon. In the lost genes analysis, we detected smelt lost 4,461 (22.16%), 2,825 (10.62%), and 1,499 (3.09%) genes compare with above three species, respectively. In the gained genes analysis, we observed smelt gain 1,133 (5.49%), 1,670 (8.09%), and 229 (1.11%) genes compare with above species, respectively. From transcriptome analysis, a total of 297 and 331 differentially expressed genes (DEGs) with False discovery rate (FDR) < 0.05 were identified in the liver and muscle tissues, respectively. Gene enrichment analysis of DEGs indicates that up-regulated genes were significantly enriched for lipid biosynthetic process (GO : 0008610, P < 0.001) and regulation of apoptotic process (GO : 0042981, P < 0.01), and down-regulated genes by immune responses such as myeloid cell differentiation (GO : 0030099, P < 0.001) in the liver under heat stress. In muscle tissue, up-regulated genes were enriched for hypoxia (GO : 0001666, P < 0.05), transcription regulator activity (GO : 0140110, P < 0.001) and calcium-release channel activity (GO : 0015278, P < 0.01), and down-regulated genes for nicotinamide nucleotide biosynthetic process (GO : 0019359, P < 0.01). The results of KEGG pathway analysis were similar to that of gene enrichment analysis. The draft genome and transcriptomic of H. nipponensis will be used as a useful genetic resource for functional and evolutionary studies. Our findings will improve understanding of the molecular mechanisms and heat responses and will be useful for predicting survival of the smelt and its closely related species under global warming.
Thomas W R Harrop, Joseph Guhlin, Gemma M McLaughlin, Elizabeth Permina, Peter Stockwell, Josh Gilligan, Marissa F Le Lec, Monica A M Gruber, Oliver Quinn, Mackenzie Lovegrove, et al.
G3 Genes|Genomes|Genetics, Volume 11; doi:10.1093/g3journal/jkab109

Abstract:
In the originally published version of this manuscript, funding information and disclosures were omitted. The following information should have been included after the Acknowledgments section.
Elizabeth Popowski, Susan J Thomson, Mareike Knäbel, Jibran Tahir, Ross N Crowhurst, Marcus Davy, Toshi M Foster, Robert J Schaffer, D Stuart Tustin, Andrew C Allan, et al.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab142

Abstract:
Commercially grown kiwifruit (genus Actinidia) are generally of two sub-species which have a base haploid genome of 29 chromosomes. The yellow-fleshed Actinidia chinensis var. chinensis, is either diploid (2n = 2x = 58) or tetraploid (2n = 4x = 116) and the green-fleshed cultivar A. chinensis var. deliciosa “Hayward,” is hexaploid (2n = 6x = 174). Advances in breeding green kiwifruit could be greatly sped up by the use of molecular resources for more efficient and faster selection, for example using marker-assisted selection (MAS). The key genetic marker that has been implemented for MAS in hexaploid kiwifruit is for gender testing. The limited marker-trait association has been reported for other polyploid kiwifruit for fruit and production traits. We have constructed a high-density linkage map for hexaploid green kiwifruit using genotyping-by-sequence (GBS). The linkage map obtained consists of 3686 and 3940 markers organized in 183 and 176 linkage groups for the female and male parents, respectively. Both parental linkage maps are co-linear with the A. chinensis “Red5” reference genome of kiwifruit. The linkage map was then used for quantitative trait locus (QTL) mapping, and successfully identified QTLs for king flower number, fruit number and weight, dry matter accumulation, and storage firmness. These are the first QTLs to be reported and discovered for complex traits in hexaploid kiwifruit.
Roland Chanet, Dorothée Baïlle, Marie-Pierre Golinelli-Cohen, Sylvie Riquier, Olivier Guittet, Michel Lepoivre, Meng-Er Huang,
G3 Genes|Genomes|Genetics, Volume 11; doi:10.1093/g3journal/jkab124

Abstract:
B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here, we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab149

Abstract:
In species with polygenic sex determination (PSD), multiple male- and female-determining loci on different proto-sex chromosomes segregate as polymorphisms within populations. The extent to which these polymorphisms are at stable equilibria is not yet resolved. Previous work demonstrated that PSD is most likely to be maintained as a stable polymorphism when the proto-sex chromosomes have opposite (sexually antagonistic) fitness effects in males and females. However, these models usually consider PSD systems with only two proto-sex chromosomes, or they do not broadly consider the dominance of the alleles under selection. To address these shortcomings, I used forward population genetic simulations to identify selection pressures that can maintain PSD under different dominance scenarios in a system with more than two proto-sex chromosomes (modeled after the house fly). I found that overdominant fitness effects of male-determining proto-Y chromosomes are more likely to maintain PSD than dominant, recessive, or additive fitness effects. The overdominant fitness effects that maintain PSD tend to have proto-Y chromosomes with sexually antagonistic effects (male-beneficial and female-detrimental). In contrast, dominant fitness effects that maintain PSD tend to have sexually antagonistic multi-chromosomal genotypes, but the individual proto-sex chromosomes do not have sexually antagonistic effects. These results demonstrate that sexual antagonism can be an emergent property of the multi-chromosome genotype without individual sexually antagonistic chromosomes. My results further illustrate how the dominance of fitness effects has consequences for both the likelihood that PSD will be maintained as well as the role sexually antagonistic selection is expected to play in maintaining the polymorphism.
, Francois Olivier Hebert, J Spencer Johnston, Richard C Hamelin, Michel Cusson, Roger C Levesque, Dawn E Gundersen-Rindal
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab150

Abstract:
The European gypsy moth, Lymantria dispar dispar (LDD), is an invasive insect and a threat to urban trees, forests and forest-related industries in North America. For use as a comparator with a previously published genome based on the LD652 pupal ovary-derived cell line, as well as whole-insect genome sequences obtained from the Asian gypsy moth subspecies L. dispar asiatica and L. dispar japonica, the whole-insect LDD genome was sequenced, assembled and annotated. The resulting assembly was 998 Mb in size, with a contig N50 of 662 Kb and a GC content of 38.8%. Long interspersed nuclear elements constitute 25.4% of the whole-insect genome, and a total of 11,901 genes predicted by automated gene finding encoded proteins exhibiting homology with reference sequences in the NCBI NR and/or UniProtKB databases at the most stringent similarity cutoff level (i.e., the gold tier). These results will be especially useful in developing a better understanding of the biology and population genetics of L. dispar and the genetic features underlying Lepidoptera in general.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab146

Abstract:
Advances in genomics techniques are expanding the range of nematode species that are amenable to transgenesis. Due to divergent codon usage biases across species, codon optimization is often a critical step for the successful expression of exogenous transgenes in nematodes. Platforms for generating DNA sequences codon-optimized for the free-living model nematode Caenorhabditis elegans are broadly available. However, until now such tools did not exist for non-Caenorhabditis nematodes. We therefore developed the Wild Worm Codon Adapter, a tool for rapid transgene codon optimization for expression in non-Caenorhabditis nematodes. The app includes built-in optimization for parasitic nematodes in the Strongyloides, Nippostrongylus, and Brugia genera as well as the predatory nematode Pristionchus pacificus. The app also supports custom optimization for any species using user-provided optimization rules. In addition, the app supports automated insertion of synthetic or native introns, as well as the analysis of codon bias in transgene and native sequences. Here, we describe this web-based tool and demonstrate how it may be used to analyze genome-wide codon bias in Strongyloides species.
, , Jason Bonnette, , David B Lowry,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab144

Abstract:
Ionomics measures elemental concentrations in biological organisms and provides a snapshot of physiology under different conditions. In this study, we evaluate genetic variation of the ionome in outbred, perennial switchgrass in three environments across the species’ native range, and explore patterns of genotype-by-environment interactions. We grew 725 clonally replicated genotypes of a large full sib family from a four-way linkage mapping population, created from deeply diverged upland and lowland switchgrass ecotypes, at three common gardens. Concentrations of 18 mineral elements were determined in whole post-anthesis tillers using ion coupled plasma mass spectrometry (ICP-MS). These measurements were used to identify quantitative trait loci (QTL) with and without QTL-by-environment interactions (QTLxE) using a multi-environment QTL mapping approach. We found that element concentrations varied significantly both within and between switchgrass ecotypes, and GxE was present at both the trait and QTL level. Concentrations of 14 of the 18 elements were under some genetic control, and 77 QTL were detected for these elements. Seventy-four percent of QTL colocalized multiple elements, half of QTL exhibited significant QTLxE, and roughly equal numbers of QTL had significant differences in magnitude and sign of their effects across environments. The switchgrass ionome is under moderate genetic control and by loci with highly variable effects across environments.
, Patrick Gonzales, Thomas J LaRocca, Yuping Song, Joanne Wuu, Michael Benatar, Björn Oskarsson, Leonard Petrucelli, Robin D Dowell, Christopher D Link, et al.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab141

Abstract:
Numerous reports have suggested that infectious agents could play a role in neurodegenerative diseases, but specific etiological agents have not been convincingly demonstrated. To search for candidate agents in an unbiased fashion, we have developed a bioinformatic pipeline that identifies microbial sequences in mammalian RNA-seq data, including sequences with no significant nucleotide similarity hits in GenBank. Effectiveness of the pipeline was tested using publicly available RNA-seq data and in a reconstruction experiment using synthetic data. We then applied this pipeline to a novel RNA-seq dataset generated from a cohort of 120 samples from amyotrophic lateral sclerosis (ALS) patients and controls, and identified sequences corresponding to known bacteria and viruses, as well as novel virus-like sequences. The presence of these novel virus-like sequences, which were identified in subsets of both patients and controls, were confirmed by quantitative RT-PCR. We believe this pipeline will be a useful tool for the identification of potential etiological agents in the many RNA-seq data sets currently being generated.
Soren Emerson, Megan Hay, Mark Smith, Ricky Granger, David Blauch, Nicole Snyder,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab143

Abstract:
The toxicity and addictive liability associated with cocaine abuse are well-known. However, its mode of action is not completely understood, and effective pharmacotherapeutic interventions remain elusive. The cholinergic effects of cocaine on acetylcholine receptors, synthetic enzymes, and degradative enzymes have been the focus of relatively little empirical investigation. Due to its genetic tractability and anatomical simplicity, the egg laying circuit of the hermaphroditic nematode, Caenorhabditis elegans, is a powerful model system to precisely examine the genetic and molecular targets of cocaine in vivo. Here, we report a novel cocaine-induced behavioral phenotype in C. elegans, cocaine-stimulated egg laying. In addition, we present the results of an in vivo candidate suppression screen of synthetic enzymes, receptors, degradative enzymes, and downstream components of the intracellular signaling cascades of the main neurotransmitter systems that control C. elegans egg laying. Our results show that cocaine-stimulated egg laying is dependent on acetylcholine synthesis and synaptic release, functional nicotinic acetylcholine receptors, and the C. elegans acetylcholinesterases.
Grant T Billings, Michael A Jones, , Amanda M Hulse-Kemp,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab145

Abstract:
Accelerated marker-assisted selection and genomic selection breeding systems require genotyping data to select the best parents for combining beneficial traits. Since 1935, the Pee Dee (PD) cotton germplasm enhancement program has developed an important genetic resource for upland cotton (Gossypium hirsutum L.), contributing alleles for improved fiber quality, agronomic performance, and genetic diversity. To date, a detailed genetic survey of the program’s eight historical breeding cycles has yet to be undertaken. The objectives of this study were to evaluate genetic diversity across and within-breeding groups, examine population structure, and contextualize these findings relative to the global upland cotton gene pool. The CottonSNP63K array was used to identify 17,441 polymorphic markers in a panel of 114 diverse PD genotypes. A subset of 4597 markers was selected to decrease marker density bias. Identity-by-state pairwise distance varied substantially, ranging from 0.55 to 0.97. Pedigree-based estimates of relatedness were not very predictive of observed genetic similarities. Few rare alleles were present, with 99.1% of SNP alleles appearing within the first four breeding cycles. Population structure analysis with principal component analysis, discriminant analysis of principal components, fastSTRUCTURE, and a phylogenetic approach revealed an admixed population with moderate substructure. A small core collection (n < 20) captured 99% of the program’s allelic diversity. Allele frequency analysis indicated potential selection signatures associated with stress resistance and fiber cell growth. The results of this study will steer future utilization of the program’s germplasm resources and aid in combining program-specific beneficial alleles and maintaining genetic diversity.
Melanie B Abrams, Claire A Dubin, Faisal AlZaben, , Pierre M Joubert, Carly V Weiss,
G3 Genes|Genomes|Genetics, Volume 11; doi:10.1093/g3journal/jkab139

Abstract:
Many familiar traits in the natural world—from lions’ manes to the longevity of bristlecone pine trees—arose in the distant past, and have long since fixed in their respective species. A key challenge in evolutionary genetics is to figure out how and why species-defining traits have come to be. We used the thermotolerance growth advantage of the yeast Saccharomyces cerevisiae over its sister species Saccharomyces paradoxus as a model for addressing these questions. Analyzing loci at which the S. cerevisiae allele promotes thermotolerance, we detected robust evidence for positive selection, including amino acid divergence between the species and conservation within S. cerevisiae populations. Because such signatures were particularly strong at the chromosome segregation gene ESP1, we used this locus as a case study for focused mechanistic follow-up. Experiments revealed that, in culture at high temperature, the S. paradoxus ESP1 allele conferred a qualitative defect in biomass accumulation and cell division relative to the S. cerevisiae allele. Only genetic divergence in the ESP1 coding region mattered phenotypically, with no functional impact detectable from the promoter. Our data support a model in which an ancient ancestor of S. cerevisiae, under selection to boost viability at high temperature, acquired amino acid variants at ESP1 and many other loci, which have been constrained since then. Complex adaptations of this type hold promise as a paradigm for interspecies genetics, especially in deeply diverged traits that may have taken millions of years to evolve.
, , , Luc Janss, , Mogens Sandø Lund
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab133

Abstract:
This work represents a novel mechanistic approach to simulate and study genomic networks with accompanying regulatory interactions and complex mechanisms of quantitative trait formation. The approach implemented in MeSCoT software is conceptually based on the omnigenic genetic model of quantitative (complex) trait, and closely imitates the basic in vivo mechanisms of quantitative trait realization. The software provides a framework to study molecular mechanisms of gene-by-gene and gene-by-environment interactions underlying quantitative trait’s realization and allows detailed mechanistic studies of impact of genetic and phenotypic variance on gene regulation. MeSCoT performs a detailed simulation of genes’ regulatory interactions for variable genomic architectures and generates complete set of transcriptional and translational data together with simulated quantitative trait values. Such data provide opportunities to study, for example, verification of novel statistical methods aiming to integrate intermediate phenotypes together with final phenotype in quantitative genetic analyses or to investigate novel approaches for exploiting gene-by-gene and gene-by-environment interactions.
Patricia P Peterson,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab134

Abstract:
Nutrient sensing is important for cell growth, aging, and longevity. In Saccharomyces cerevisiae, Sch9, an AGC-family protein kinase, is a major nutrient sensing kinase homologous to mammalian Akt and S6 kinase. Sch9 integrates environmental cues with cell growth by functioning downstream of TORC1 and in parallel with the Ras/PKA pathway. Mutations in SCH9 lead to reduced cell growth in dextrose medium; however, reports on the ability of sch9Δ mutants to utilize non-fermentable carbon sources are inconsistent. Here, we show that sch9Δ mutant strains cannot grow on non-fermentable carbon sources and rapidly accumulate suppressor mutations, which reverse growth defects of sch9Δ mutants. sch9Δ induces gene expression of three transcription factors required for utilization of non-fermentable carbon sources, Cat8, Adr1, and Hap4, while sch9Δ suppressor mutations, termed sns1 and sns2, strongly decrease the gene expression of those transcription factors. Despite the genetic suppression interactions, both sch9Δ and sns1 (or sns2) homozygous mutants have severe defects in meiosis. By screening mutants defective in sporulation, we identified additional sch9Δ suppressor mutants with mutations in GPB1, GPB2, and MCK1. Using library complementation and genetic analysis, we identified SNS1 and SNS2 to be IRA2 and IRA1, respectively. Furthermore, we discovered that lifespan extension in sch9Δ mutants is dependent on IRA2 and that PKA inactivation greatly increases basal expression of CAT8, ADR1, and HAP4. Our results demonstrate that sch9Δ leads to complete loss of growth on non-fermentable carbon sources and mutations in MCK1 or genes encoding negative regulators of the Ras/PKA pathway reverse sch9Δ mutant phenotypes.
Minqi Li, , Xiaoxiao Dong, ,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab138

Abstract:
Transposons can create allelic diversity that affects gene expression and phenotypic diversity. The detection of transposon polymorphisms at a genome-wide scale across a large population is difficult. Here, we developed a targeted sequencing approach to monitor transposon polymorphisms of interest. This approach can interrogate the presence or absence of transposons reliably across various genotypes. Using this approach, we genotyped a set of 965 transposon-related presence/absence polymorphisms in a diverse panel of 16 maize (Zea mays L.) inbred lines that are representative of the major maize breeding groups. About 70% of the selected regions can be effectively assayed in each genotype. The consistency between the capture-based assay and PCR-based assay are 98.6% based on analysis of 24 randomly selected transposon polymorphisms. By integrating the transposon polymorphisms data with gene expression data, ∼18% of the assayed transposon polymorphisms were found to be associated with variable gene expression levels. A detailed analysis of 18 polymorphisms in a larger association panel confirmed the effects of 10 polymorphisms, with one of them having a stronger association with expression than nearby SNP markers. The effects of seven polymorphisms were tested using a luciferase-based expression assay, and one was confirmed. Together, this study demonstrates that the targeted sequencing assay is an effective way to explore transposon function in a high-throughput manner.
Tetsuro Kawano-Sugaya, Koji Yatsu, Tsuyoshi Sekizuka, Kentaro Itokawa, Masanori Hashino, Rina Tanaka, Makoto Kuroda
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab126

Abstract:
Summary Many of software for network visualization are available, but existing software have not been optimized to infection cluster visualization, especially the current worldwide invasion of COVID-19 since 2019. To reach the spatiotemporal understanding of epidemics, we have developed Haplotype Explorer. In Haplotype Explorer, users can explore the network interactively with metadata like accession number, locations, and collection dates. Time dependent transition of the network can be exported as continuous sections for making a movie. Here, we introduce features and products of Haplotype Explorer, demonstrating time-dependent snapshots and a movie of haplotype networks inferred from total of 4,282 SARS-CoV-2 genomes. Abstract The worldwide eruption of COVID-19 that began in Wuhan, China in late 2019 reached 10 million cases by late June 2020. In order to understand the epidemiological landscape of the COVID-19 pandemic, many studies have attempted to elucidate phylogenetic relationships between collected viral genome sequences using haplotype networks. However, currently available applications for network visualization are not suited to understand the COVID-19 epidemic spatiotemporally due to functional limitations, that motivated us to develop Haplotype Explorer, an intuitive tool for visualizing and exploring haplotype networks. Haplotype Explorer enables to dissect epidemiological consequences via interactive node filters and provides the perspective on infectious disease dynamics depend on regions and time, such as introduction, outbreak, expansion, and containment. Here, we demonstrate the effectiveness of Haplotype Explorer by showing features and an example of visualization. The demo using SARS-CoV-2 genomes are available at https://github.com/TKSjp/HaplotypeExplorer/blob/master/Example/. There are several examples using SARS-CoV-2 genomes and Dengue virus serotype 1 E-genes sequence.
, Andrew N Gale, Anna Bailey, Kali Barnes, Kiersten Colotti, Michal Mass, Luke B Morina, Bailey Robertson, Remy Schwab, Niki Tselepidakis, et al.
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab137

Abstract:
We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.
Anna L Tyler, Baha El Kassaby, Georgi Kolishovski, Jake Emerson, Ann E Wells, J Matthew Mahoney,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab131

Abstract:
It is well understood that variation in relatedness among individuals, or kinship, can lead to false genetic associations. Multiple methods have been developed to adjust for kinship while maintaining power to detect true associations. However, relatively unstudied are the effects of kinship on genetic interaction test statistics. Here, we performed a survey of kinship effects on studies of six commonly used mouse populations. We measured inflation of main effect test statistics, genetic interaction test statistics, and interaction test statistics reparametrized by the Combined Analysis of Pleiotropy and Epistasis (CAPE). We also performed linear mixed model (LMM) kinship corrections using two types of kinship matrix: an overall kinship matrix calculated from the full set of genotyped markers, and a reduced kinship matrix, which left out markers on the chromosome(s) being tested. We found that test statistic inflation varied across populations and was driven largely by linkage disequilibrium. In contrast, there was no observable inflation in the genetic interaction test statistics. CAPE statistics were inflated at a level in between that of the main effects and the interaction effects. The overall kinship matrix overcorrected the inflation of main effect statistics relative to the reduced kinship matrix. The two types of kinship matrices had similar effects on the interaction statistics and CAPE statistics, although the overall kinship matrix trended toward a more severe correction. In conclusion, we recommend using an LMM kinship correction for both main effects and genetic interactions and further recommend that the kinship matrix be calculated from a reduced set of markers in which the chromosomes being tested are omitted from the calculation. This is particularly important in populations with substantial population structure, such as recombinant inbred lines in which genomic replicates are used.
Tijana Cvetković, Fabiola Areces-Berazain, Damien D Hinsinger, Daniel C Thomas, Jan J Wieringa, Santhana K Ganesan,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab136

Abstract:
Malvaceae s.l., the most diverse family within Malvales, includes well-known species of great economic importance like cotton, cacao, and durian. Despite numerous phylogenetic analyses employing multiple markers, relationships between several of its nine subfamilies, particularly within the largest lineage/Malvadendrina, remain unclear. In this study, we attempted to resolve the relationships within the major clades of Malvaceae s.l. using plastid genomes of 48 accessions representing all subfamilies. Maximum likelihood and Bayesian analyses recovered a fully resolved and well-supported topology confirming the split of the family into/Byttneriina (/Grewioideae +/Byttnerioideae) and/Malvadendrina. Within/Malvadendrina,/Helicteroideae occupied the earliest branching position, followed by/Sterculioideae./Brownlowioideae,/Tiliodeae, and/Dombeyoideae formed a clade sister to/Malvatheca (/Malvoideae +/Bombacoideae), a grouping morphologically supported by the lack of androgynophore. Results from dating analyses suggest that all subfamilies originated during hot or warm phases in the Late Cretaceous to Paleocene. This study presents a well-supported phylogenetic framework for Malvaceae s.l. that will aid downstream revisions and evolutionary studies of this economically important plant family.
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