All Title Author
Keywords Abstract

PLOS ONE  2012 

Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)

DOI: 10.1371/journal.pone.0041570

Full-Text   Cite this paper   Add to My Lib


Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M2 mutants in a common wheat cultivar ‘Jinmai 47’. Numerous phenotypes with altered morphological and agronomic traits were observed from the M2 and M3 lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.


[1]  Chua NH, Tingey SV (2006) Plant biotechnology: Looking forward to the next ten years. Curr Opin Biotechnol 17: 103–104.
[2]  Dong C, Dalton-Morgan J, Vincent K, Sharp P (2009) A Modified TILLING Method for Wheat Breeding. Plant Gen 2: 39–47.
[3]  International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436(7052): 793–800.
[4]  Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, et al. (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449(7161): 463–467.
[5]  Eversole K (2009) Advancements towards sequencing the bread wheat genome: An update of the projects of the international wheat genome sequencing consortium. Annual Wheat Newsletter 55: In press.
[6]  Minoia S, Petrozza A, D'Onofrio O, Piron F, Mosca G, et al. (2010) A new mutant genetic resource for tomato crop improvement by TILLING technology. BMC Research Notes 3: 69.
[7]  Hirochika H (2001) Contribution of the Tos17 retrotransposon to rice functional genomics. Curr Opin Plant Biol 4: 118–122.
[8]  Weigel D, Ahn JH, Blázquez MA, Borevitz JO, Christensen SK, et al. (2000) Activation tagging in Arabidopsis. Plant Physiol 122: 1003–1013.
[9]  Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657.
[10]  Jeon J, Lee S, Jung KH, Jun SH, Jeong DH, et al. (2000) T-DNA insertional mutagenesis for functional genomics in rice. Plant J 22: 561–570.
[11]  Parry MAJ, Madgwick PJ, Bayon C, Tearall K, Hernandez-Lopez A, et al. (2009) Mutation discovery for crop improvement. Journal of Experimental Botany 60: 2817–2825.
[12]  Uauy C, Paraiso F, Colasuonno P, Tran RK, Tsai H, et al. (2009) A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC plant Biology 9: 115.
[13]  Fu D, Uauy C, Blechl A, Dubcovsky J (2007) RNA interference for wheat functional gene analysis. Transgenic Res 16: 689–701.
[14]  Fox JL (2004) Monsanto cuts GM wheat. Nat Biotechnol 22: 645.
[15]  Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23: 75–81.
[16]  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.
[17]  Henikoff S, Till BJ, Comai L (2004) TILLING, traditional mutagenesis meets functional genomics. Plant Physiology 135(2): 630–636.
[18]  Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, et al. (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164: 731–740.
[19]  Till BJ, Cooper J, Tai TH, Colowit P, Greene EA, et al. (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7: 19.
[20]  Till BJ, Comai L, Henikoff S (2007) Tilling and EcoTILLING for crop improvement. In: Varshney RK, Tuberosa R, editors. Genomics-Assisted Crop Improvement. Genomics Approaches and Platforms. Netherlands: Springer. pp. 333–349.
[21]  Sabetta W, Alba V, Blanco A, Montemurro C (2011) SunTILL: a TILLING resource for gene function analysis in sunflower. Plant Methods 7: 20.
[22]  Rothe N (2010) Validation of tilling populations in diploid and hexaploid wheat. M.S. thesis. Kansass State University. Available:
[23]  Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, et al. (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 4: 12.
[24]  Caldwell DG, McCallum N, Shaw P, Muehlbauer GJ, Marshall DF, et al. (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J 40(1): 143–150.
[25]  Talamè V, Bovina R, Sanguineti MC, Tuberosa R, Lundqvist U, et al. (2008) TILLMore, a resource for the discovery of chemically induced mutants in barley. Plant Biotechnol J 6(5): 477–485.
[26]  Gottwald S, Bauer P, Komatsuda T, Lundqvist U, Stein N (2009) TILLING in the two-rowed barley cultivar ‘Barke’ reveals preferred sites of functional diversity in the gene HvHox1. BMC Research Notes 2: 258.
[27]  Wu JL, Wu C, Lei C, Baraoidan M, Bordeos A, et al. (2005) Chemical-and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics. Plant Mol Biol 59: 85–97.
[28]  Xin Z, Wang ML, Barkley NA, Burow G, Franks C, et al. (2008) Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population. BMC Plant Biology 8: 103.
[29]  Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, et al. (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8(1): 9.
[30]  Muth J, Hartje S, Twyman RM, Hofferbert HR, Tacke E, et al. (2008) Precision breeding for novel starch variants in potato. Plant Biotechnology J 6: 576–584.
[31]  Knoll JE, Ramos ML, Zeng Y, Holbrook CC, Chow M, et al. (2011) TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.). BMC Plant Biology 11: 81.
[32]  Wang N, Wang Y, Tian F, King GJ, Zhang C, et al. (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(4): 751–765.
[33]  Stephenson P, Baker D, Girin T, Perez A, Amoah S, et al. (2010) A rich TILLING resource for studying gene function in Brassica rapa. BMC Plant Biology 10: 62.
[34]  Till BJ, Colbert T, Tompa R, Enns LC, Codomo CA, et al. (2003) High-throughput TILLING for functional genomies. Methords Mol Biol 236: 205–220.
[35]  Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, et al. (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics 279: 213–223.
[36]  Raghavan C, Naredo MEB, Wang HH, Atienza G, Liu B, et al. (2007) Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping. Mol Breeding 19: 87–101.
[37]  Oleykowski CA, Mullins CRB, Godwin AK, Yeung AT (1998) Mutation detection using a novel plant endonuclease. Nucleic Acids Research 26: 4597–4602.
[38]  Bentley A, MacLennan B, Calvo J, Dearolf CR (2000) Targeted recovery of mutations in Drosophila. Genetics 156(3): 1169–1173.
[39]  Piron F, Nicola? M, Mino?a S, Piednoir E, Moretti A, et al. (2010) An Induced Mutation in Tomato eIF4E Leads to Immunity to Two Potyviruses. PLoS ONE 5(6): e11313.
[40]  Dahmani-Mardas F, Troadec C, Boualem A, Lévêque S, Alsadon AA, et al. (2010) Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach. PLoS ONE 5(12): e15776.
[41]  Chawade A, Sikora P, Br?utigam M, Larsson M, Vivekanand V, et al. (2010) Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes. BMC Plant Biology 10: 86.
[42]  Nomura T, Ishihara A, Yanagita RC, Endo TR, Iwamura H (2005) Three genomes differentially contribute to the biosynthesis of benzoxazinones in hexaploid wheat. Proc Natl Acad Sci USA 102(45): 16490–16495.
[43]  Chen L, Wang SQ, Hu YG (2011) Detection of SNPs in the VRN-A1 gene of common wheat (Triticum aestivum L.) by a modified Ecotilling method using agarose gel electrophoresis. Australian Journal of Crop Science 5(3): 321–329.
[44]  Halloran GM, Pennell AL (1982) Duration and rate of development phases in wheat in two environments. Ann Bot 49: 115–121.
[45]  Slafer GA, Rawson HM (1996) Responses to photoperiod change with phenophase and temperature during wheat development. Field Crops Res 46: 1–13.
[46]  González FG, Slafer GA, Miralles DJ (2005) Pre-anthesis development and number of fertile florets in wheat as affected by photoperiod sensitivity genes Ppd-D1 and Ppd-B1. Euphytica 146: 253–269.
[47]  Aldrich C (1993) CTAB DNA extraction from plant tissues. Plant Mol Biol Rep 11: 128–141.
[48]  Song W, Henry RJ (1995) Molecular analysis of the DNA polymorphism of wild barley (Hordeum spontaneum) germplasm using the polymerase chain reaction. Genetic Resources and Crop Evolution 42: 273–281.
[49]  Przetakiewicz J, Nadolaka-orczy A, Orczyk W (2002) The use of RAPD and semi-random markers to verify somatic hybrids between diploid lines of Solanum tuberosum L. Cellular & Molecular Biology Letters 7: 671–676.
[50]  Naghavi MR, Mardi M, Ramshini HA, Fazelinasab B (2004) Comparative analyses of the genetic diversity among bread wheat genotypes based on RAPD and SSR markers. Iranian Journal of Biotechnology 2(3): 195–202.
[51]  Bibi S, Dahot MU, Khan IA, Khatri A, Naqvi MH (2009) Study of genetic diversity in wheat (Triticum Aestivum L.) using random amplified polymorphic DNA (RAPD) markers. Pak J Bot 41(3): 1023–1027.
[52]  Merril CR, Harrington M, Alley V (1984) A photodevelopment silver stain for the rapid visualization of proteins separated on polyacrylamide gels. Electrophoresis 5: 289–297.
[53]  Bassam BJ, Caetano-Anollés G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry 196: 80–83.


comments powered by Disqus