Update on TILLING in Grass Species TILLING in Grass Species

The reverse genetics detection of single base changes in genes of interest, whether induced or endogenous, is an extremely powerful tool for functional genomics. Two major thrusts of research in the grasses are characterizing the function of all the genes in various genomes and then comparing those functions among species. Targeting Induced Local Lesions IN Genomes (TILLING) has proven to be a valuable methodology for finding these polymorphisms in a wide range of plant and animal species and providing mutants carrying them to the researcher. With the advent of NextGen sequencing, targeted evaluation of thousands of genes across mutagenized populations with thousands of individuals will becomemuchmore widely available, and for a wide range of grass species. Completed genome sequences from multiple grass species greatly increase the power of reverse genetic approaches to understanding gene function. Perhaps the greatest advantage is the ability to triangulate gene functions among multiple taxa such as rice (Oryza sativa), maize (Zea mays), and sorghum (Sorghum bicolor), and, together with efforts to create transposon and deletion collections in these plants, reverse genetics analysis of functionally important single nucleotide polymorphisms (SNPs) and mutations is a crucial part of the grass functional genomics toolkit. Transposon and T-DNA approaches can be used in some species, but they are less applicable to most others, particularly grasses, where agroinfection needs to be coerced. Instead, chemical mutagenesis and TILLING have come into wide use (Comai and Henikoff, 2006), particularly for the grasses domesticated as crops. In 2002, there was a single TILLING facility in Seattle, a year later several more, and now there are over a dozen worldwide, both public and private. This marked increase in the number of TILLING projects under way, the mention of these methods and resources in grant proposals, and statements of future directions in various research communities and even the availability of commercial kits are, perhaps, the strongest pieces of evidence that the reverse genetics of point mutations is now a fixture in the genomics landscape. At the core of each of these efforts are the creation, increase, maintenance, and distribution of large, mutagenized populations. With the exception of maize, where pollen mutagenesis is possible, these populations have been developed through seed treatment, primarily with ethyl methane sulfonate (EMS) but occasionally with the use of other chemicals such as sodium azide and/or methylnitrosourea (MNU) in species where EMS proves less effective. Typically, the approach has been to try a range of treatment severity and select the treatment that gives a desired amount (typically 30%–40%) of M2 families segregating for embryo and seedling lethality (Till et al., 2006). Where TILLING is already established for a species, new mutagenized populations can also be assessed directly by TILLING a sampling of five to 10 genes. These mutant populations have also proven to be valuable resources for forward genetic screening as well, with research communities taking advantage of the growouts for seed increase by various TILLING projects to walk fields looking for novel phenotypes of interest. In maize, mutants with altered recombination frequency (C. Weil, H. Dooner, W. Eggleston, S. Stack, and P. Schnable, unpublished data), inflorescence architecture mutants (R. Schmidt, unpublished data), mutants with altered starch digestion rates (Groth et al., 2008), and defective kernel mutants identified in forward screens of the TILLING populations are just some of those under investigation. The TILLING populations are also being screened as a whole for photosynthetic mutants and cold tolerance mutants (J. Langdale and P. Rivera, unpublished data). As originally designed (McCallum et al., 2000), the methodology of TILLING screens pooled DNA samples taken from individuals within a mutagenized population; 8-fold pools have proven to be the most generally robust. Using informatics tools such as CODDLe (www.proweb.org/coddle/) that compare the sequence of interest against the known database for similarities, an approximately 1,500-bp region of the target sequence is identified that has the highest likelihood of producing mutations that will have detrimental effects on the gene. These predictions are based on the ability of a mutation to create either a nonsense codon, an alteration of a transcript splicing site, or a nonconservative missense mutation in a highly conserved domain of the predicted protein. The selected region (or any alternative that can be hand designated by a user of the TILLING services) is then PCR amplified from each of the pooled DNAs and screened enzymatically for the presence of mismatched bases that result if one member of a pool 1 This work was supported by the National Science Foundation Plant Genome Award (grant no. DBI0604765) and the Purdue Agricultural Research Station. * E-mail [email protected]. The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Clifford F. Weil ([email protected]). www.plantphysiol.org/cgi/doi/10.1104/pp.108.128785