Mutagenomics: A Rapid, High-Throughput Method to Identify Causative Mutations from a Genetic Screen

编号 040027301

推送时间 20210111

研究领域 森林培育 

年份 2021 

类型 期刊 

语种 英语

标题 Mutagenomics: A Rapid, High-Throughput Method to Identify Causative Mutations from a Genetic Screen

来源期刊 Plant Physiology

期 第273期

发表时间 20200904

关键词 mutagenomics;  genetic screen;  arabidopsis;  HY5;  cytokinin; 

摘要 Genetic screens are powerful tools to dissect complex biological processes, but a rate-limiting step is often the cloning of targeted genes. Here, we present a strategy, “mutagenomics,” to identify causal mutations from a screen in a high throughput fashion in the absence of backcrossing. Mutagenomics is initiated by sequencing the genomes of the mutants identified, which are then subjected to a three-stage pipeline. The first stage identifies sequence changes in genes previously linked to the targeted pathway. The second stage uses heuristics derived from a simulation strategy to identify genes that are represented by multiple independent alleles more often than expected by chance. The third stage identifies candidate genes for the remaining lines by sequencing multiple lines of common descent. Our simulations indicate that sequencing as few as three to four sibling lines generally results in fewer than five candidate genes. We applied mutagenomics to a screen for Arabidopsis (Arabidopsis thaliana) mutants involved in the response to the phytohormone cytokinin. Mutagenomics identified likely causative genes for many of the mutant lines analyzed from this screen, including 13 alleles of the gene encoding the ARABIDOPSIS HIS KINASE4 cytokinin receptor. The screen also identified 1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE7, an ACC synthase homolog involved in ethylene biosynthesis, and ELONGATED HYPOCOTYL5 (HY5), a master transcriptional regulator of photomorphogenesis. HY5 was found to mediate a subset of the transcriptional response to cytokinin. Mutagenomics has the potential to accelerate the pace and utility of genetic screens in Arabidopsis.

服务人员 孙小满

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