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A T-DNA mutant screen that combines high-throughput phenotyping with the efficient identification of mutated genes by targeted genome sequencing.

ABSTRACT: BACKGROUND:Nitrogen dioxide (NO2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana. A high-throughput mutant screen was established to identify genes involved in this type of programmed cell death. RESULTS:Altogether 14,282 lines of SALK T-DNA insertion mutants were screened. Growing 1000 pooled mutant lines per tray and simultaneous NO2 fumigation of 4 trays in parallel facilitated high-throughput screening. Candidate mutants were selected based on visible symptoms. Sensitive mutants showed lesions already after fumigation for 1?h with 10?ppm (ppm) NO2 whereas tolerant mutants were hardly damaged even after treatment with 30?ppm NO2. Identification of T-DNA insertion sites by adapter ligation-mediated PCR turned out to be successful but rather time consuming. Therefore, next generation sequencing after T-DNA-specific target enrichment was tested as an alternative screening method. The targeted genome sequencing was highly efficient due to (1.) combination of the pooled DNA from 124 candidate mutants in only two libraries, (2.) successful target enrichment using T-DNA border-specific 70mer probes, and (3.) stringent filtering of the sequencing reads. Seventy mutated genes were identified by at least 3 sequencing reads. Ten corresponding mutants were re-screened of which 8 mutants exhibited NO2-sensitivity or -tolerance confirming that the screen yielded reliable results. Identified candidate genes had published functions in HR, pathogen resistance, and stomata regulation. CONCLUSIONS:The presented NO2 dead-or-alive screen combined with next-generation sequencing after T-DNA-specific target enrichment was highly efficient. Two researchers finished the screen within 3?months. Moreover, the target enrichment approach was cost-saving because of the limited number of DNA libraries and sequencing runs required. The experimental design can be easily adapted to other screening approaches e.g. involving high-throughput treatments with abiotic stressors or phytohormones.

SUBMITTER: Frank U

PROVIDER: S-EPMC6894221 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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A T-DNA mutant screen that combines high-throughput phenotyping with the efficient identification of mutated genes by targeted genome sequencing.

Frank Ulrike U Kublik Susanne S Mayer Dörte D Engel Marion M Schloter Michael M Durner Jörg J Gaupels Frank F

BMC plant biology 20191204 1

<h4>Background</h4>Nitrogen dioxide (NO<sub>2</sub>) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana. A high-throughput mutant screen was established to identify genes involved in this type of programmed cell death.<h4>Results</h4>Altogether 14,282 lines of SALK T-DNA insertion mutants were screened. Growing 1000 pooled mutant lines per tray and simultaneous NO<sub>2</sub> fumigation of 4 trays in parallel facilitated high-throughput screening. Candidate mutants wer ...[more]

PMID: 31801481

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Project description:BackgroundIntellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation.MethodsWe report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases.ResultsWe identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders.ConclusionsWith a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism.

Dataset Information

A T-DNA mutant screen that combines high-throughput phenotyping with the efficient identification of mutated genes by targeted genome sequencing.

Publications

A T-DNA mutant screen that combines high-throughput phenotyping with the efficient identification of mutated genes by targeted genome sequencing.

Similar Datasets

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