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.
At a glance
- Tomato Genome Consortium. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485, 635–641 (2012).
- Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing. Plant J. 80, 136–148 (2014). et al.
- The genome of the stress-tolerant wild tomato species Solanum pennellii. Nat. Genet. 46, 1034–1038 (2014). et al.
- Genomic analyses provide insights into the history of tomato breeding. Nat. Genet. 46, 1220–1226 (2014). et al.
- Genome-wide analysis of intraspecific DNA polymorphism in 'Micro-Tom', a model cultivar of tomato (Solanum lycopersicum). Plant Cell Physiol. 55, 445–454 (2014). et al.
- An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141, 1147–1162 (1995). &
- In silico screening of a saturated mutation library of tomato. Plant J. 38, 861–872 (2004). , , , &
- A new model system for tomato genetics. Plant J. 12, 1465–1472 (1997). et al.
- A high throughput system for transposon tagging and promoter trapping in tomato. Plant J. 38, 861–872 (2000). , , , & et al.
- Tomato mutants as tools for functional genomics. Curr. Opin. Plant Biol. 5, 112–117 (2002). &
- TOMATOMA: a novel tomato mutant database distributing Micro-Tom mutant collections. Plant Cell Physiol. 52, 283–296 (2011). et al.
- Convergence of developmental mutants into a single tomato model system: ′Micro-Tom′ as an effective toolkit for plant development research. Plant Methods 7, 18 (2011). et al.
- Biochemical and histological characterization of tomato mutants. An. Acad. Bras. Cienc. 84, 573–585 (2012). et al.
- Micro-Tom mutants for functional analysis of target genes and discovery of new alleles in tomato. Plant Biotechnol. 30, 225–231 (2013). et al.
- Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss. Plant J. 60, 363–377 (2009). et al.
- Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato. Plant Physiol. 159, 945–960 (2012). et al.
- The identification of cutin synthase: formation of the plant polyester cutin. Nat. Chem. Biol. 8, 609–611 (2012). et al.
- Inhibition of CUTIN DEFICIENT 2 causes defects in cuticle function and structure and metabolite changes in tomato fruit. Plant Cell Physiol. 54, 1535–1548 (2013). et al.
- The tomato SlSHINE3 transcription factor regulates fruit cuticle formation and epidermal patterning. New Phytol. 197, 468–480 (2013). et al.
- Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin-abundant mutants and a new hypomorphic allele of GDSL lipase. Plant Physiol. 164, 888–906 (2014). et al.
- An alternative pathway to beta-carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato. Proc. Natl. Acad. Sci. USA 97, 11102–11107 (2000). , , &
- Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of beta-carotene and xanthophylls in plants. Plant Cell 14, 333–342 (2002). , , &
- Manipulation of phytoene levels in tomato fruit: effects on isoprenoids, plastids, and intermediary metabolism. Plant Cell 19, 3194–3211 (2007). et al.
- Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J. 53, 717–730 (2008). , , , &
- Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum). Plant J. 79, 453–465 (2014). et al.
- The tomato mutation nxd1 reveals a gene necessary for neoxanthin biosynthesis and demonstrates that violaxanthin is a sufficient precursor for abscisic acid biosynthesis. Plant J. 78, 80–93 (2014). , , , &
- Zooming in on a quantitative trait for tomato yield using interspecific introgressions. Science 305, 1786–1789 (2004). , , , &
- Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement. Nat. Biotechnol. 24, 447–454 (2006). et al.
- Gene and metabolite regulatory network analysis of early developing fruit tissues highlights new candidate genes for the control of tomato fruit composition and development. Plant Physiol. 149, 1505–1528 (2009). et al.
- Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes. Science 341, 175–179 (2013). et al.
- Genome-wide association in tomato reveals 44 candidate loci for fruit metabolic traits. Plant Physiol. 165, 1120–1132 (2014). et al.
- Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit. Plant J. 81, 24–39 (2015). et al.
- The tomato Blind gene encodes a MYB transcription factor that controls the formation of lateral meristems. Proc. Natl. Acad. Sci. USA 99, 1064–1069 (2002). et al.
- The flowering gene SINGLE FLOWER TRUSS drives heterosis for yield in tomato. Nat. Genet. 42, 459–463 (2010). , &
- Shoot branching and leaf dissection in tomato are regulated by homologous gene modules. Plant Cell 23, 3595–3609 (2011). et al.
- Role of tomato BRANCHED1-like genes in the control of shoot branching. Plant J. 67, 701–714 (2011). et al.
- Synchronization of the flowering transition by the tomato TERMINATING FLOWER gene. Nat. Genet. 44, 1393–1398 (2012). et al.
- Optimization of crop productivity in tomato using induced mutations in the florigen pathway. Nat. Genet. 46, 1337–1342 (2014). et al.
- A role for APETALA1/fruitfull transcription factors in tomato leaf development. Plant Cell 25, 2070–2083 (2013). et al.
- Evolutionary developmental transcriptomics reveals a gene network module regulating interspecific diversity in plant leaf shape. Proc. Natl. Acad. Sci. USA 111, 2616–2621 (2014). et al.
- Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nat. Genet. 39, 787–791 (2007). et al.
- MicroRNA regulation of plant innate immune receptors. Proc. Natl. Acad. Sci. USA 109, 1790–1795 (2012). et al.
- Overdominant quantitative trait loci for yield and fitness in tomato. Proc. Natl. Acad. Sci. USA 103, 12981–12986 (2006). et al.
- The role of induced mutations in world food security. In Induced Plant Mutations in the Genomics Era (ed. Shu, Q.Y.) 33–38 (Food and Agricultural Organization of the United Nations, Rome, 2009). &
- Single-nucleotide mutations for plant functional genomics. Annu. Rev. Plant Biol. 54, 375–401 (2003). &
- A new mutant genetic resource for tomato crop improvement by TILLING technology. BMC Res. Notes 3, 69 (2010). et al.
- Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries. Plant Cell Physiol. 52, 1994–2005 (2011). et al.
- NEATTILL: a simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications. Plant Methods 6, 3 (2010). et al.
- TILLING identification of ascorbate biosynthesis tomato mutants for investigating vitamin C in tomato. Plant Biotechnol. 30, 309–314 (2013). et al.
- Failure of the tomato trans-acting short interfering RNA program to regulate AUXIN RESPONSE FACTOR3 and ARF4 underlies the wiry leaf syndrome. Plant Cell 24, 3575–3589 (2012). et al.
- Using next-generation sequencing to isolate mutant genes from forward genetic screens. Nat. Rev. Genet. 15, 662–676 (2014).
- SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat. Methods 6, 550–551 (2009). et al.
- Next-generation mapping of Arabidopsis genes. Plant J. 67, 715–725 (2011). et al.
- Genome sequencing reveals agronomically important loci in rice using MutMap. Nat. Biotechnol. 30, 174–178 (2012). et al.
- MutMap+: genetic mapping and mutant identification without crossing in rice. PLoS One 8, e68529 (2013). et al.
- MutMap-Gap: whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii. New Phytol. 200, 276–283 (2013). et al.
- MutMap accelerates breeding of a salt-tolerant rice cultivar. Nat. Biotechnol. 33, 445–449 (2015). et al.
- Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato. Plant Biotechnol. J. 14, 51–60 (2015). , , , &
- SNP discovery and linkage map construction in cultivated tomato. DNA Res. 17, 381–391 (2010). et al.
- Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009). &
- The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). et al.
- The glycerol-3-phosphate acyltransferase GPAT6 from tomato plays a central role in fruit cutin biosynthesis. Plant Physiol. 171, 894–913 (2016). et al.
- An induced mutation in tomato eIF4E leads to immunity to two potyviruses. PLoS One 5, e11313 (2010). et al.
- Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening. Mol. Breed. 29, 801–812 (2012). et al.
- Genome-wide SNP genotyping to infer the effects on gene functions in tomato. DNA Res. 20, 221–233 (2013). et al.
- Genetic and physiological characterization of tomato cv. Micro-Tom. J. Exp. Bot. 57, 2037–2047 (2006). , , , &
- Whole genome resequencing in tomato reveals variation associated with introgression and breeding events. BMC Genomics 14, 791 (2013). et al.
- Retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit. Science 319, 5869 (2008). , , , &
- SNP genotyping using KASPar assays. Methods Mol. Biol. 1245, 243–256 (2015). &
- BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9, 253 (2008). et al.
- Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry. Sex Plant Reprod. 15, 311–320 (2003). , , , &
- Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011). et al.
- Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013). , &
- Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010). et al.
- Galaxy: a web-based genome analysis tool for experimentalists. Curr. Protoc. Mol. Biol. Chapter 19 Unit 19.10.1-21 (2010).
- Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 15, 1451–1455 (2005). et al.
- Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat. Protoc. 1, 387–396 (2006). , , , &
- A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6, 80–92 (2012). et al.
- Micro-Tom. A Miniature Dwarf Tomato 1–6 (Florida Agricultural Experimental Station, 1989). &
- Culture of the tomato Micro-Tom cultivar in Greenhouse. Methods Mol. Biol. 1363, 57–64 (2016). et al.
- Flexible tools for gene expression and silencing in tomato. Plant Physiol. 151, 1729–1740 (2009). et al.
- microRNA156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development. Plant J. 78, 604–618 (2014). et al.
- Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 166, 1292–1297 (2014). , , &
- Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol. 166, 455–469 (2014). et al.
- A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation. Plant Physiol. 150, 1122–1134 (2009). et al.
- Coupling virus-induced gene silencing to exogenous green fluorescence protein expression provides a highly efficient system for functional genomics in Arabidopsis and across all stages of tomato fruit development. Plant Physiol. 156, 1278–1291 (2011). et al.
- Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nat. Protoc. 9, 1549–1562 (2014). &
- Supplementary Software (4,378 KB)
The ‘compare_WT_mutant_samtools_vcf_v5.py’ script.
- Supplementary Table 1 (42 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.