Targeting‐induced local lesions in genomes (TILLING) is a powerful reverse‐genetics tool that enables high‐throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis‐based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)‐mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

中文翻译：

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用于栽培烟草的高效 TILLING 平台。


靶向基因组中诱发的局部病变 (TILLING) 是一种强大的反向遗传学工具，可以对植物中的基因组变异进行高通量筛选。尽管 TILLING 已针对许多二倍体植物开发，但由于其基因组复杂性，该技术已在极少数多倍体物种中使用。在这里，我们使用甲磺酸乙酯 (EMS) 诱变的 1,536 个个体群体，为异源四倍体栽培烟草 ( Nicotiana tabacum L.) 建立了一个基于毛细管电泳的高效 TILLING 平台。我们优化了核酸内切酶制备、叶组织取样、DNA 提取、标准化、合并、PCR 扩增、异源双链体形成和毛细管电泳的程序。在使用 7 个目标基因和 8 个 PCR 片段的测试筛选中，我们获得了 118 个突变体。突变密度估计约为平均每 106 kb 1 个突变。表型分析表明，两个重金属转运蛋白基因HMA2S和HMA4T的突变导致镉和锌的积累减少，这一点通过 CRISPR/Cas9 生成敲除突变体得到独立证实。我们的结果表明，这个强大的 TILLING 平台（可在 http://www.croptilling.org 上获取）可用于烟草，以促进功能基因组学应用。