Rapid identification of heterozygous mutations in <i>Drosophila melanogaster</i> using genomic capture sequencing

Wang, Hui; Chattopadhyay, Abanti; Li, Zhe; Daines, Bryce; Li, Yumei; Gao, Chunying; Gibbs, Richard A.; Zhang, Kun; Chen, Rui

doi:10.1101/gr.102921.109

Cited by 21 publications

(20 citation statements)

References 31 publications

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“…Previous use of NGS in targeted gene cloning has focused on model organisms [ e.g. , A. thaliana (Mokry et al 2011; Uchida et al 2011; Austin et al 2011), Drosophila melanogaster (Blumenstiel et al 2009; Wang et al 2010), Caenorhabditis elegans (Sarin et al 2008), and fission yeast (Irvine et al 2009)]. Sorghum, while sequenced (Paterson et al 2009), has not been used successfully to clone genes using NGS tools.…”

Section: Discussionmentioning

confidence: 99%

Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

Krothapalli

Buescher

et al. 2013

Genetics

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Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of next-generation sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, which encodes a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of (1) a sequenced genome, (2) a strongly penetrant and easily measurable phenotype, and (3) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the postgenomic era.

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Section: Discussionmentioning

confidence: 99%

Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

Krothapalli

Buescher

et al. 2013

Genetics

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“…Unlike most other mutagens, the molecular lesions caused by EMS are essentially random, ensuring that most genes of interest will be targeted and that multiple lesions will be found in each gene. Whole genome sequencing now allow us to reliably and efficiently map EMS induced lesions at very reasonable costs (Blumenstiel et al, 2009;Wang et al, 2010) (H.J.B., unpublished data).…”

Section: : Forward Genetic Approaches To Isolate Novel Neuronal Genesmentioning

confidence: 99%

Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly

Venken

Simpson

Bellen

2011

Neuron

406

340

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Research in the fruit fly Drosophila melanogaster has lead to insights in neural development, axon guidance, ion channel function, synaptic transmission, learning and memory, diurnal rythmicity, and neural disease that have had broad implications for neuroscience. Drosophila is currently the eukaryotic model organism that permits the most sophisticated in vivo manipulations to address the function of neurons and neuronally expressed genes. Here, we summarize many of the techniques that help assess the role of specific neurons by labeling, removing, or altering their activity. We also survey genetic manipulations to identify and characterize neural genes by mutation, over-expression, and protein labeling. Here, we attempt to acquaint the reader with available options and contexts to apply these methods.

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“…In parallel with its medical applications, targeted capture began to emerge as a powerful approach to address evolutionary questions in humans (Briggs et al 2009; Burbano et al 2010; Krause et al 2010; Li et al 2010; Yi et al 2010). Although initially restricted to species with sequenced genomes (e.g., chimpanzee, Perry et al 2010; Drosophila melanogaster , Wang et al 2010; maize, Fu et al 2010), more recent extensions to non-reference species have demonstrated the potential to effectively use targeted capture across diverse taxa (Cosart et al 2011; Mason et al 2011; Vallender 2011; Bi et al , 2012, 2013; Good et al 2015). Nonetheless, targeted capture remains relatively uncommon in evolutionary biology studies.…”

Section: Introductionmentioning

confidence: 99%

Targeted capture in evolutionary and ecological genomics

2015

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The rapid expansion of next-generation sequencing has yielded a powerful array of tools to address fundamental biological questions at a scale that was inconceivable just a few years ago. Various genome partitioning strategies to sequence select subsets of the genome have emerged as powerful alternatives to whole genome sequencing in ecological and evolutionary genomic studies. High throughput targeted capture is one such strategy that involves the parallel enrichment of pre-selected genomic regions of interest. The growing use of targeted capture demonstrates its potential power to address a range of research questions, yet these approaches have yet to expand broadly across labs focused on evolutionary and ecological genomics. In part, the use of targeted capture has been hindered by the logistics of capture design and implementation in species without established reference genomes. Here we aim to 1) increase the accessibility of targeted capture to researchers working in non-model taxa by discussing capture methods that circumvent the need of a reference genome, 2) highlight the evolutionary and ecological applications where this approach is emerging as a powerful sequencing strategy, and 3) discuss the future of targeted capture and other genome partitioning approaches in light of the increasing accessibility of whole genome sequencing. Given the practical advantages and increasing feasibility of high-throughput targeted capture, we anticipate an ongoing expansion of capture-based approaches in evolutionary and ecological research, synergistic with an expansion of whole genome sequencing.

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Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing

Cited by 21 publications

References 31 publications

Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly

Targeted capture in evolutionary and ecological genomics

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