Department of Biology, University of Texas at Arlington, Arlington, TX, 76019, USA.
Heredity (Edinb). 2021 Dec;127(6):535-545. doi: 10.1038/s41437-021-00478-x. Epub 2021 Oct 19.
Forward genetic screening using the alkylating mutagen ethyl methanesulfonate (EMS) is an effective method for identifying phenotypic mutants of interest, which can be further genetically dissected to pinpoint the causal genetic mutations. An accurate estimate of the rate of EMS-induced heritable mutations is fundamental for determining the mutant sample size of a screening experiment that aims to saturate all the genes in a genome with mutations. This study examines the genome-wide EMS-induced heritable base-substitutions in three species of the freshwater microcrustacean Daphnia to help guide screening experiments. Our results show that the 10 mM EMS treatment induces base substitutions at an average rate of 1.17 × 10/site/generation across the three species, whereas a significantly higher average mutation rate of 1.75 × 10 occurs at 25 mM. The mutation spectrum of EMS-induced base substitutions at both concentration is dominated by G:C to A:T transitions. Furthermore, we find that female Daphnia exposed to EMS (F individuals) can asexually produce unique mutant offspring (F) for at least 3 consecutive broods, suggestive of multiple broods as F mutants. Lastly, we estimate that about 750 Fs are needed for all genes in the Daphnia genome to be mutated at least once with a 95% probability. We also recommend 4-5 Fs should be collected from each F mutant through sibling crossing so that all induced mutations could appear in the homozygous state in the F population at 70-80% probability.
正向遗传筛选使用烷化诱变剂甲基甲磺酸乙酯（EMS）是鉴定感兴趣的表型突变体的有效方法，可以进一步进行遗传分析，以确定导致遗传突变的原因。准确估计 EMS 诱导的可遗传突变率对于确定筛选实验中的突变体样本大小至关重要，筛选实验旨在使基因组中的所有基因都发生突变。本研究检查了三种淡水枝角类动物的全基因组 EMS 诱导的可遗传碱基替换，以帮助指导筛选实验。我们的结果表明，10 mM EMS 处理在三个物种中平均以 1.17×10 个/位点/代的速度诱导碱基替换，而在 25 mM 时，平均突变率显著更高，为 1.75×10。在这两种浓度下，EMS 诱导的碱基替换的突变谱主要由 G:C 到 A:T 转换组成。此外，我们发现暴露于 EMS 的雌性枝角类动物（F 个体）可以无性繁殖出独特的突变后代（F）至少连续三代，这表明 F 突变体可能有多个后代。最后，我们估计至少需要 750 个 Fs 才能使 Daphnia 基因组中的所有基因至少发生一次突变，概率为 95%。我们还建议从每个 F 突变体通过同胞交配收集 4-5 个 Fs，以便在 F 群体中，所有诱导的突变都以 70-80%的概率出现在纯合状态。
