For instance, several PCR-based methods such as the adapter ligation-mediated PCR rely on insert-specific primers to selectively amplify the T-DNA/genomic DNA junction before identification of the mutated gene by sequencing. The fact that the T-DNA sequence is known facilitates convenient mapping of the insertion site. For Arabidopsis, the SALK collection represents the largest source of T-DNA insertion mutants covering almost the complete genome. Thousands of mutant lines were generated in Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Medicago truncatula, and other plant species by insertional mutagenesis of genes using retrotransposons or Agrobacterium tumefaciens-derived T-DNA. involving high-throughput treatments with abiotic stressors or phytohormones.įorward genetics by mutant screens is a widely used approach to experimentally assign biological functions to genes or characterize physiological processes through the identification of involved genes. The experimental design can be easily adapted to other screening approaches e.g. Moreover, the target enrichment approach was cost-saving because of the limited number of DNA libraries and sequencing runs required. Two researchers finished the screen within 3 months. The presented NO 2 dead-or-alive screen combined with next-generation sequencing after T-DNA-specific target enrichment was highly efficient. Identified candidate genes had published functions in HR, pathogen resistance, and stomata regulation. Ten corresponding mutants were re-screened of which 8 mutants exhibited NO 2-sensitivity or -tolerance confirming that the screen yielded reliable results. Seventy mutated genes were identified by at least 3 sequencing reads. 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. Therefore, next generation sequencing after T-DNA-specific target enrichment was tested as an alternative screening method. Identification of T-DNA insertion sites by adapter ligation-mediated PCR turned out to be successful but rather time consuming. Sensitive mutants showed lesions already after fumigation for 1 h with 10 ppm (ppm) NO 2 whereas tolerant mutants were hardly damaged even after treatment with 30 ppm NO 2. Candidate mutants were selected based on visible symptoms. Growing 1000 pooled mutant lines per tray and simultaneous NO 2 fumigation of 4 trays in parallel facilitated high-throughput screening. ResultsĪltogether 14,282 lines of SALK T-DNA insertion mutants were screened. A high-throughput mutant screen was established to identify genes involved in this type of programmed cell death. Nitrogen dioxide (NO 2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana.
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