G3 Genes|Genomes|Genetics

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ISSN / EISSN : 2160-1836 / 2160-1836
Current Publisher: Oxford University Press (OUP) (10.1093)
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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.
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 an 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.
Daniel Mariyappa, Arthur Luhur, Danielle Overton,
G3 Genes|Genomes|Genetics; doi:10.1093/g3journal/jkab161

Abstract:
The generation of Drosophila stable cell lines have 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 the 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.
Vera Pavese, Emile Cavalet Giorsa, Lorenzo Barchi, , Daniela Torello Marinoni, Ezio Portis, Stuart James Lucas, 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 analysed with BlastP and InterProScan software. To characterise ‘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.
Louise van der Weyden, 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 make them attractive therapeutic targets, we performed a CRISPR activation screen using a library of guide RNAs (gRNAs) targeting the transcription start sites of 2,195 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-characterised 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 colonisation 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; 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 1,021 Mbp, composed of 2,553 scaffolds and it has an N50 of 1,105 kbp. 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 (dsx, IMP, PSI) 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, Yasuhiro Matsuo, 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 play 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, Mark Cooper
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.
, 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.
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