Abstract
TILLING (Targeting Induced Local Lesions IN Genomes) is a well-known reverse genetics technique designed to detect unknown SNPs (single nucleotide polymorphisms) in genes of interest using an enzymatic digestion and is widely employed in plant and animal genomics. The main advantage of this technique is that it allows for the high-throughput identification of an allelic series of mutants with a range of modified functions for a particular gene. In this chapter, we aim to give a detailed introduction of how to establish a TILLING platform for identifying mutants in plants, including generation of a large mutant population, DNA and seed library preparation, mutation identification based on a LI-COR4300 DNA analyzer, and confirmation of functions of the mutated genes.
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References
McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeted screening for induced mutations. Nat Biotechnol 18:455–457
McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol 123: 439–442
Till BJ, Colbert T, Tompa R, Enns LC, Codomo CA, Johnson JE, Reynolds SH, Henikoff JG, Greene EA, Steine MN, Comai L, Henikoff S (2003) High-throughput TILLING for functional genomics. Methods Mol Biol 236:205–220
Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, Henikoff S (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13:524–530
Jones MO, Piron-Prunier F, Marcel F, Piednoir-Barbeau E, Alsadon AA, Wahb-Allah MA, Al-Doss AA, Bowler C, Bramley PM, Fraser PD, Bendahmane A (2012) Characterisation of alleles of tomato light signalling genes generated by TILLING. Phytochemistry 79:78–86
Gady AL, Vriezen WH, Van de Wal MH, Huang P, Bovy AG, Visser RG, Bachem CW (2012) Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening. Mol Breed 29:801–812
Chen L, Huang L, Min D, Phillips A, Wang S, Madgwick PJ, Parry MA, Hu YG (2012) Development and characterization of a new TILLING population of common bread wheat (Triticum aestivum L.). PLoS One 7:e41570
Sabetta W, Alba V, Blanco A, Montemurro C (2011) sunTILL: a TILLING resource for gene function analysis in sunflower. Plant Methods 7:20
Slade AJ, McGuire C, Loeffler D, Mullenberg J, Skinner W, Fazio G, Holm A, Brandt KM, Steine MN, Goodstal JF, Knauf VC (2012) Development of high amylose wheat through TILLING. BMC Plant Biol 12:69
Sikora P, Chawade A, Larsson M, Olsson J, Olsson O (2012) Mutagenesis as a tool in plant genetics, functional genomics, and breeding. Int J Plant Genomics 2011:314829
Okabe Y, Asamizu E, Saito T, Matsukura C, Ariizumi T, Bres C, Rothan C, Mizoguchi T, Ezura H (2011) 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
Knoll JE, Ramos ML, Zeng Y, Holbrook CC, Chow M, Chen S, Maleki S, Bhattacharya A, Ozias-Akins P (2011) TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.). BMC Plant Biol 11:81
Stephenson P, Baker D, Girin T, Perez A, Amoah S, King GJ, Ostergaard L (2010) A rich TILLING resource for studying gene function in Brassica rapa. BMC Plant Biol 10:62
Minoia S, Petrozza A, D'Onofrio O, Piron F, Mosca G, Sozio G, Cellini F, Bendahmane A, Carriero F (2010) A new mutant genetic resource for tomato crop improvement by TILLING technology. BMC Res Notes 3:69
Fitzgerald TL, Kazan K, Li Z, Morell MK, Manners JM (2010) A high-throughput method for the detection of homologous gene deletions in hexaploid wheat. BMC Plant Biol 10:264
Uauy C, Paraiso F, Colasuonno P, Tran RK, Tsai H, Berardi S, Comai L, Dubcovsky J (2009) A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 9:115
Perry J, Brachmann A, Welham T, Binder A, Charpentier M, Groth M, Haage K, Markmann K, Wang TL, Parniske M (2009) TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements. Plant Physiol 151: 1281–1291
Morita R, Kusaba M, Iida S, Yamaguchi H, Nishio T, Nishimura M (2009) Molecular characterization of mutations induced by gamma irradiation in rice. Genes Genet Syst 84:361–370
Le Signor C, Savois V, Aubert G, Verdier J, Nicolas M, Pagny G, Moussy F, Sanchez M, Baker D, Clarke J, Thompson R (2009) Optimizing TILLING populations for reverse genetics in Medicago truncatula. Plant Biotechnol J 7:430–441
Elias R, Till BJ, Mba C, Al-Safadi B (2009) Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC Res Notes 2:141
de Lorenzo L, Merchan F, Laporte P, Thompson R, Clarke J, Sousa C, Crespi M (2009) A novel plant leucine-rich repeat receptor kinase regulates the response of Medicago truncatula roots to salt stress. Plant Cell 21:668–680
Wang N, Wang Y, Tian F, King GJ, Zhang C, Long Y, Shi L, Meng J (2008) A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING. New Phytol 180:751–765
Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K, Nagato Y, Kurata N (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics 279:213–223
Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, El-Mellouki T, Liu S, Ritchie R, Nielsen N, Bilyeu KD, Meksem K, Comai L, Henikoff S (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8:9
Till BJ, Cooper J, Tai TH, Colowit P, Greene EA, Henikoff S, Comai L (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7:19
Horst I, Welham T, Kelly S, Kaneko T, Sato S, Tabata S, Parniske M, Wang TL (2007) TILLING mutants of Lotus japonicus reveal that nitrogen assimilation and fixation can occur in the absence of nodule-enhanced sucrose synthase. Plant Physiol 144:806–820
Heckmann AB, Lombardo F, Miwa H, Perry JA, Bunnewell S, Parniske M, Wang TL, Downie JA (2006) Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume. Plant Physiol 142:1739–1750
Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, Young K, Bowers E, Codomo CA, Enns LC, Odden AR, Greene EA, Comai L, Henikoff S (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 4:12
Perry JA, Wang TL, Welham TJ, Gardner S, Pike JM, Yoshida S, Parniske M (2003) A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol 131:866–871
Tsai H, Howell T, Nitcher R, Missirian V, Watson B, Ngo KJ, Lieberman M, Fass J, Uauy C, Tran RK, Khan AA, Filkov V, Tai TH, Dubcovsky J, Comai L (2011) Discovery of rare mutations in populations: TILLING by sequencing. Plant Physiol 156:1257–1268
Kurowska M, Daszkowska-Golec A, Gruszka D, Marzec M, Szurman M, Szarejko I, Maluszynski M (2011) TILLING: a shortcut in functional genomics. J Appl Genet 52:371–390
Weil CF (2009) TILLING in grass species. Plant Physiol 149:158–164
Colbert T, Till BJ, Tompa R, Reynolds S, Steine MN, Yeung AT, McCallum CM, Comai L, Henikoff S. (2001) High- throughput screening for induced point mutations. Plant Physiol 126:480–484
Oleyowski CA, Bronson Mullins CR, Godwin AK, Yeung AT (1998) Mutation detection using a novel plant endonuclease. Nucleic Acids Res 26:4597–4602
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Wang, N., Shi, L. (2015). Screening of Mutations by TILLING in Plants. In: Batley, J. (eds) Plant Genotyping. Methods in Molecular Biology, vol 1245. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1966-6_15
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DOI: https://doi.org/10.1007/978-1-4939-1966-6_15
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