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

编号 040022101

推送时间 20200113

研究领域 森林培育 

年份 2020 

类型 期刊 

语种 英语

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

来源期刊 BMC Plant Biology

期 第221期

发表时间 20191204

关键词 Arabidopsis thaliana;  T-DNA insertion;  Mutant screen;  High-throughput phenotyping;  Target enrichment; 
Next generation sequencing;  Adapter ligation-mediated PCR; 

摘要 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.

服务人员 孙小满

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