An efficient TILLING platform for cultivated tobacco

Area of Science:

• Plant Genomics
• Functional Genomics
• Crop Science

Background:

• Targeting-Induced Local Lesions in Genomes (TILLING) is a valuable reverse-genetics tool for screening plant genomic variations.
• Its application in polyploid species is limited due to genomic complexity.

Purpose of the Study:

• To establish an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.).
• To facilitate high-throughput screening of induced mutations in tobacco for functional genomics studies.

Main Methods:

• Utilized an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 tobacco individuals.
• Optimized procedures for DNA extraction, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis.
• Screened seven target genes, identifying 118 mutants with an average mutation density of 1 per 106 kb.

Main Results:

• Successfully established a TILLING platform for polyploid tobacco.
• Identified mutations in heavy metal transporter genes (HMA2S and HMA4T) affecting cadmium and zinc accumulation.
• Validated findings using CRISPR/Cas9 gene editing.

Conclusions:

• The developed TILLING platform is effective for tobacco functional genomics.
• This technology can accelerate the discovery of gene functions in polyploid crops.
• The platform is accessible at http://www.croptilling.org.