Abstract
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.
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Claims 4
Claim · model-written summary
The tobacco TILLING screen yielded 118 mutants across seven target genes.
Verbatim
e0“In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants.”
Claim · model-written summary
Mutation density averaged approximately one mutation per 106 kb.
- Taxon
- tobacco
Nicotiana tabacumt0 - Outcome
- mutation densitye0
- Value
- one mutation per 106 kbe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The mutation density was estimated to be approximately one mutation per 106 kb on average.”
t0“cultivated tobacco”
Claim · model-written summary
HMA2S mutation reduced cadmium and zinc accumulation in tobacco.
- Taxon
- tobaccoNicotiana tabacumt0
- Treatment
- mutationse0
- Gene
- HMA2Se0
- Outcome
- accumulation of cadmium and zince0
- Direction
- reducede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc”
t0“cultivated tobacco”
Claim · model-written summary
HMA4T mutation reduced cadmium and zinc accumulation in tobacco.
- Taxon
- tobaccoNicotiana tabacumt0
- Treatment
- mutationse0
- Gene
- HMA4Te0
- Outcome
- accumulation of cadmium and zince0
- Direction
- reducede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc”
t0“cultivated tobacco”
The same questions in other papers
loss of function · molecular: metabolites · Nicotiana tabacum (tobacco)Positive and negative results
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- positive
- negative
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