Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing.
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 F2population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2segregants 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.
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
V
Garcia
Institut National de la Recherche Agronomique and Université de Bordeaux, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
Cécile
http://orcid.org/0000-0001-6460-4637
C
Bres
Institut National de la Recherche Agronomique and Université de Bordeaux, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
Daniel
D
Just
Institut National de la Recherche Agronomique and Université de Bordeaux, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
Lucie
L
Fernandez
Institut National de la Recherche Agronomique and Université de Bordeaux, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
Fabienne Wong Jun
FW
Tai
Institut National de la Recherche Agronomique and Université de Bordeaux, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
Jean-Philippe
JP
Mauxion
Institut National de la Recherche Agronomique US1279 Etude du Polymorphisme des Génomes Végétaux, CEA-Institut de Génomique-CNG, Evry, France.
Marie-Christine
MC
Le Paslier
Institut National de la Recherche Agronomique US1279 Etude du Polymorphisme des Génomes Végétaux, CEA-Institut de Génomique-CNG, Evry, France.
Aurélie
A
Bérard
Institut National de la Recherche Agronomique US1279 Etude du Polymorphisme des Génomes Végétaux, CEA-Institut de Génomique-CNG, Evry, France.
Dominique
D
Brunel
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
Koh
K
Aoki
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany.
Saleh
S
Alseekh
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany.
Alisdair R
AR
Fernie
School of Biological Sciences, Royal Holloway University of London, Egham, UK.
Paul D
PD
Fraser
Institut National de la Recherche Agronomique and Université de Bordeaux, Unité Mixte de Recherche 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
Christophe
http://orcid.org/0000-0002-6831-2823
C
Rothan
D004252:DNA Mutational Analysis / Q000379:methods*; D005020:Ethyl Methanesulfonate / Q000378:metabolism*; D014644:Genetic Variation; D018551:Solanum lycopersicum / Q000235:genetics*; D009154:Mutation; D013997:Time Factors
D005020:Ethyl Methanesulfonate