Mutagenomics: A Rapid, High-Throughput Method to Identify Causative Mutations from a Genetic Screen
Mutagenomics: A Rapid, High-Throughput Method to Identify Causative Mutations from a Genetic Screen
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DOI：
10.1104/pp.20.00609
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发表时间：
2020-12-01
期刊：
影响因子：
7.4
通讯作者：
Kieber, Joseph J.
中科院分区：
文献类型：
--
作者：
Hodgens, Charles;Chang, Nicole;Kieber, Joseph J.
开发了一种用于高通量识别对应于在遗传筛查中识别的突变的致病基因的方法，并将其应用于细胞分裂素突变体的筛查。基因筛查是剖析复杂生物过程的强大工具，但一个速度限制步骤往往是目标基因的克隆。在这里，我们提出了一种策略，“突变基因组学”，在没有回交的情况下，以高通量的方式从筛查中识别原因突变。突变基因组学是通过对已鉴定的突变体的基因组进行测序启动的，然后经过三个阶段的管道。第一阶段确定先前与靶向途径相关联的基因的序列变化。第二阶段使用源自模拟策略的启发式方法来识别由多个独立等位基因代表的基因，其频率比预期的要高。第三阶段通过对多个共同血统的品系进行测序，确定剩余品系的候选基因。我们的模拟表明，只要对三到四个兄弟姐妹系进行测序，通常会产生不到五个候选基因。我们应用突变基因组学来筛选与植物激素细胞分裂素反应有关的拟南芥突变体。突变基因组学确定了从该筛查分析的许多突变系的可能致病基因，包括编码拟南芥HIS KINASE 4细胞分裂素受体的基因的13个等位基因。该筛选还鉴定了参与乙烯生物合成的ACC合成酶同源物1-氨基环丙烷-1-羧酸酯(ACC)SYNTHase 7和光形态建成的主要转录调节因子拉长的HYPOCOTYL5(HY5)。HY5被发现介导了对细胞分裂素的转录反应的一部分。突变基因组学有可能加快拟南芥遗传筛选的步伐和用途。
A method for the high-throughput identification of causative genes corresponding to mutations identified in a genetic screen is developed and applied to a screen for cytokinin mutants. 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.