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Targeting Induced Local Lesions IN Genomes

TILLING (Targeting Induced Local Lesions IN Genomes) is a general reverse genetic technique that uses traditional chemical mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high-throughput screens for the discovery of mutations. We are based at the UC Davis Genome Center and provide a TILLING service for rice, hexaploid and tetraploid wheat, Arabidopsis thaliana, and tomato.

Background

The explosion of DNA sequence information has created a unique opportunity to investigate the function of genes. The approach that determines the function of genes first defined by DNA sequence analysis is referred to as reverse genetics. The tools available for reverse genetics of plants include the use of transposons or T-DNA for gene tagging and the use of RNA interference. While powerful, these methods have limitations. For example, they do not work in all plant species.

Targeting Induced Local Lesions IN Genomes (TILLING) is reliable and widely applicable. TILLING combines chemical mutagenesis with mutation screens of pooled PCR products, resulting in the isolation of missense and nonsense mutant alleles of the targeted genes. TILLING has two significant advantages over existing plant gene knock-out tools: first, it is applicable to any plant since it does not require transgenic or cell culture manipulations. Second, it produces an allelic series of mutations including hypomorphic alleles that are useful for genetic analysis. The TILLING technology can be used to discover and survey natural variation. This method is called Ecotilling and has been applied to Arabidopsis and human (see Publications).

History

TILLING was originally conceived by Claire McCallum, a UW graduate student who wanted targeted mutations in the chromomethylase genes of Arabidopsis. Chuck Dearolf and collaborators independently conceived a similar project for Drosophila. TILLING in Seattle was developed in collaboration with the Henikoff lab which led to the Seattle TILLING Project (STP), a FHCRC-UW collaboration that provided Arabidopsis and fruit fly mutants. With the departure of Luca Comai from Seattle (Summer 2006), STP is now operated by Steve Henikoff. The TILLING objectives have been split: diploid Arabidopsis and fruit flies remained at STP, while the rice project moved to Davis. In June 2011 the Henikoff lab closed STP and the Arabidopsis DNA is being transferred to UCD where a diploid Arabidopsis TILLING will be offered.

Overview of TILLING at the UCD Genome Center

Rice TILLING at UCD is carried out in collaboration with the laboratory of Tom Tai, USDA-ARS at Davis. Rice Tilling was funded by USDA-NRI and the Rockefeller Foundation and is presently sustained on user's fees. Wheat TILLING has been developed at UCD in collaboration with Jorge Dubcovsky at the UCD department of Plant Sciences and is funded by a USDA-NRI award. Tomato TILLING is in collaboration with Roger Chetelat and Allan VanDeynze at UC Davis and is funded by a USDA-NRI plant genome award to provide a service to the tomato community. Arabidopsis TILLING We currently offer a tetraploid population of Arabidopsis. This population provides a novel approach to isolate knock-out mutants in genes that are required in the gametophyte, and has been developed in collaboration with the labs of Sundaresan Venkatesan (Sundar) and Simon Chan. The STP diploid population of Arabidopsisis is being transfered to Davis from Seattle and will be offered in the close future. Starting in the fall of 2007, we have converted to TILLING by Sequencing and essentially abandoned the LiCor-CELI mismatch detection method. Research to develop the TILLING_by_Sequencing method using rice, wheat, and arabidopsis as a model system is now funded by an award from the NSF Plant Genome Program. Our progress is summarized in a paper in Plant Physiology. Methods and data from this work are available on the TILLING_by_Sequencing page.

Results

• Rice. A Nipponbare population of over 4000 individuals yields about 4 mutations/Mb of diploid DNA. See examples in our Rice results page
• Tetraploid arabidopsis. A tetraploid Col-0 population of 528 individuals yields about 19 mutations/Mb of diploid DNA. See examples in our AT4X page
• Tomato
• Wheat

Publications

^{[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]}

1. ↑ Tsai H et al. (2013) Production of a High-Efficiency TILLING Population through Polyploidization. Plant Physiol 161: 1604-14 PubMed
2. ↑ Missirian V et al. (2011) Statistical Mutation Calling from Sequenced Overlapping DNA Pools in TILLING Experiments. BMC Bioinformatics 12: 287 PubMed
3. ↑ Tsai H et al. (2011) Discovery of rare mutations in populations: TILLING by sequencing. Plant Physiol ': PubMed
4. ↑ Uauy C et al. (2009) A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 9: 115 PubMed
5. ↑ Cooper JL et al. (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8: 9 PubMed
6. ↑ Till BJ et al. (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7: 19 PubMed
7. ↑ Comai L & Henikoff S (2006) TILLING: practical single-nucleotide mutation discovery. Plant J 45: 684-94 PubMed
8. ↑ Till BJ et al. (2006) High-throughput TILLING for Arabidopsis. Methods Mol Biol 323: 127-35 PubMed
9. ↑ Till BJ et al. (2006) A protocol for TILLING and Ecotilling in plants and animals. Nat Protoc 1: 2465-77 PubMed
10. ↑ Enns LC et al. (2005) Two callose synthases, GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility. Plant Mol Biol 58: 333-49 PubMed
11. ↑ Till BJ et al. (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 4: 12 PubMed
12. ↑ Henikoff S et al. (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol 135: 630-6 PubMed
13. ↑ Till BJ et al. (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res 32: 2632-41 PubMed
14. ↑ Comai L et al. (2004) Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 37: 778-86 PubMed
15. ↑ Greene EA et al. (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164: 731-40 PubMed
16. ↑ Till BJ et al. (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13: 524-30 PubMed
17. ↑ Till BJ et al. (2003) High-throughput TILLING for functional genomics. Methods Mol Biol 236: 205-20 PubMed
18. ↑ McCallum CM et al. (2000) Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol 123: 439-42 PubMed
19. ↑ McCallum CM et al. (2000) Targeted screening for induced mutations. Nat Biotechnol 18: 455-7 PubMed

Funding

Funding for Rice and Tomato TILLING and the forthcoming service is by USDA Cooperative State Research, Education, and Extension Service (CSREES), NRI Plant Genome Program and by a grant from the Rockefeller Foundation. TILLING-by-Sequencing is funded by the NSF Plant Genome Research Program.