RRC ID 47757
Author Garcia V, Bres C, Just D, Fernandez L, Tai FW, Mauxion JP, Le Paslier MC, Bérard A, Brunel D, Aoki K, Alseekh S, Fernie AR, Fraser PD, Rothan C.
Title Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing.
Journal Nat Protoc
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.
Volume 11(12)
Pages 2401-2418
Published 2016-12-1
DOI 10.1038/nprot.2016.143
PII nprot.2016.143
PMID 27809315
MeSH DNA Mutational Analysis / methods* Ethyl Methanesulfonate / metabolism* Genetic Variation Lycopersicon esculentum / genetics* Mutation* Time Factors
IF 11.334
Times Cited 23
WOS Category BIOCHEMICAL RESEARCH METHODS
Resource
Tomato TOMJPF00001