The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6–12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.
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This work was supported by the CEA-IG/CNG for conducting QC of DNA and Illumina sequencing. We thank A. Boland, M.T. Bihoreau and their staff. We are grateful to the Genotoul Toulouse Midi-Pyrenees bioinformatics platform and the Sigenae group (specially S. Maman) for providing help, as well as computing and storage resources. This project was funded by grants from INRA AIP Bioressources and the ERANET project 'TomQML'. F.W.J.T. was supported by a grant from ANR Bioadapt project 'Adaptom'.
The authors declare no competing financial interests.
The ‘compare_WT_mutant_samtools_vcf_v5.py’ script. (ZIP 4 kb)
Typical carotenoid content found in ripe fruit (Breaker+7) from yellow mutant as compared with the WT background (Micro-Tom line). Separations were performed by UPLC-PDA and quantitative determinations from dose response curves. FW: Fresh Weight. (PDF 41 kb)
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Garcia, V., Bres, C., Just, D. et al. Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing. Nat Protoc 11, 2401–2418 (2016). https://doi.org/10.1038/nprot.2016.143
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