Abstract

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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Acknowledgements

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'.

Author information

Author notes

    • Virginie Garcia
    • , Cécile Bres
    • , Daniel Just
    •  & Lucie Fernandez

    These authors contributed equally to this work.

Affiliations

  1. Institut National de la Recherche Agronomique and Université de Bordeaux, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.

    • Virginie Garcia
    • , Cécile Bres
    • , Daniel Just
    • , Lucie Fernandez
    • , Fabienne Wong Jun Tai
    • , Jean-Philippe Mauxion
    •  & Christophe Rothan
  2. Institut National de la Recherche Agronomique US1279 Etude du Polymorphisme des Génomes Végétaux, CEA-Institut de Génomique-CNG, Evry, France.

    • Marie-Christine Le Paslier
    • , Aurélie Bérard
    •  & Dominique Brunel
  3. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.

    • Koh Aoki
  4. Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany.

    • Saleh Alseekh
    •  & Alisdair R Fernie
  5. School of Biological Sciences, Royal Holloway University of London, Egham, UK.

    • Paul D Fraser

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Contributions

C.B., D.J., L.F., V.G. and C.R. developed the original protocol. D.B., J.-P.M. and A.B. performed the sequencing experiments. F.W.J.T. performed computational analyses. C.B., L.F., D.J., F.W.J.T., M.-C.L.P., K.A., S.A., A.R.F., P.D.F. and C.R. contributed sections to the manuscript. C.B., L.F., D.J. and C.R. collated and standardized the text. All authors read and approved the final version of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Cécile Bres.

Supplementary information

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  1. 1.

    Supplementary Software

    The ‘compare_WT_mutant_samtools_vcf_v5.py’ script.

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  1. 1.

    Supplementary Table 1

    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.

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DOI

https://doi.org/10.1038/nprot.2016.143

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