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J Integr Plant Biol, 2020, 62 (2): 165-180, Research Article
An efficient TILLING platform for cultivated tobacco
Yu-Long Gao1†, Xue-Feng Yao2,3†, Wen-Zheng Li1, Zhong-Bang Song1, Bing-Wu Wang1, Yu-Ping Wu1, Jun-Li Shi1, Guan-Shan Liu4, Yong-Ping Li1 and Chun-Ming Liu2,5*
1Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Yunnan Academy of Tobacco
Agricultural Sciences, Kunming 650021, China
2Key Laboratory of Plant Molecular Physiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China
3The University of Chinese Academy of Sciences, Beijing 100049, China
4Tobacco Research Institute, the Chinese Agriculture Academy of Sciences, Qingdao 266101, China
5Institute of Crop Sciences, the Chinese Academy of Agricultural Sciences, Beijing 100081, China

These authors contributed equally to this work.
Email: Chun-Ming Liu (
doi: 10.1111/jipb.12784

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 can be used in tobacco to facilitate functional genomics applications.

Received: 24 October 2018      Accepted:    Online on:
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