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 become much more 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 carries a mutation that the other members do not. Heteroduplex molecules that form at the last heating and reannealing step of the amplification can be cleaved at any mismatch by S1-family nucleases such as Cel1, found in crude celery (Apium graveolens) juice extracts (Till et al., 2004a). The cleaved products are then resolved, either by HPLC or in sequence analyzers (slab gel or capillary format), and sized. The pooling strategy allows identification of the particular mutant individual within a pool. After reamplifying the target from specific, putative mutants, it is sequenced to identify the specific base that has been altered. Information about the mutation (its position, predicted effect on the protein, and stock number for seed of the line carrying the mutation) is then returned to users of TILLING for further study and characterization. In contrast to typical forward mutagenesis screens, TILLING populations aim for the maximum number of mutations per plant that still allow the plants to be viable and fertile. Depending on the mutation density within the population being screened (a rate of one mutation per 500 kb or less is regarded as serviceable) and the random distribution of individual mutations throughout the genome of a given mutant population, TILLING can return allelic series for specific genes, with a wide variety of phenotypic effects. Certain types of mutations (for example, those that are gametophytic lethals and thus are not transmitted) simply cannot be recovered, but a major advantage to these populations is that most lethal and sterile alleles are maintained as heterozygotes and can still be assessed. In addition, the range of severity for point mutations means that, oftentimes, sublethal alleles of essential genes and substerile alleles of fertility genes can be analyzed as part of an allelic series. For maize, of 576 mutations delivered to date, informatics (e.g. SIFT [www.proweb.org]) based on nonconservative substitutions in conserved sequence motifs suggest that 14% are damaging alleles; however, 258 (or approximately 45%) of the mutations overall are nonsilent, and it is important to emphasize that any of these have the potential to show a biologically interesting phenotype. EcoTILLING gel image of switchgrass. Using primers designed against the maize Argonaute-like gene ago111, 48 accessions of switchgrass were compared to the upland variety Cave-in-Rock. Two duplicate sets of samples are loaded, and mismatches (SNPs) between the reference genome and the variety tested appear as bands indicated by a matching pair of squares in both duplicates. For simplicity, only the LI-COR image from the 800-nm channel is shown. The black bar at the left indicates that the entire 1,401-bp target is within exon sequence; size standards are in base pairs. Discovery of induced point mutations in maize genes by TILLING Maize haplotype variation as revealed by EcoTILLING. PVP, Plant variety protection; these lines are the recently off-patent inbreds LH1, LH123Ht, LH82, PH207, PHG35, PHG39, PHG47, PHG84, PHJ40, and PHZ51. MDL, Maize diversity lines (Liu et al., 2003). Discovery of induced point mutations in maize genes by TILLING Maize haplotype variation as revealed by EcoTILLING. PVP, Plant variety protection; these lines are the recently off-patent inbreds LH1, LH123Ht, LH82, PH207, PHG35, PHG39, PHG47, PHG84, PHJ40, and PHZ51. MDL, Maize diversity lines (Liu et al., 2003). TILLING projects among major grass crop species (maize, barley [Hordeum vulgare], rice, wheat [Triticum aestivum]) have been in operation for several years (Table II), using a variety of mutagens, and the reader is referred to these project Web pages for detailed information about each project. Existing and proposed TILLING projects in grass species Existing and proposed TILLING projects in grass species TILLING was designed, initially, as a cost-effective means of screening populations for changes in individual genes. At the time, sequencing costs and throughput were such that it was important to have a preliminary screening tool to identify the individuals that carried a mutation before sequencing efforts on that gene were carried out. This generation of sequencing technologies, which produce gigabases of data at extremely low cost, has changed that landscape considerably. Now, simply resequencing the gene of interest in thousands of lines is both feasible and inexpensive, particularly for mutagenized inbred lines where the unmutagenized inbred sequence can serve as a scaffold for sequence assembly. Resequencing entire genomes across a mutagenized population that numbers in the thousands is still not cost effective, and is debatable even once the goal of a $1,000 genome sequence is achieved. However, targeted resequencing of hundreds, or even thousands, of interesting genes within these populations is now within our grasp, cost effective, and can be done for as many as 30 to 50 genes per instrument run, potentially exceeding 2,000 genes analyzed per year per instrument. Various TILLING and SNP discovery projects are moving toward these sequencing approaches, using all three of the major platforms currently available (Roche 454, Illumina/Solexa, and ABI SOLiD; Comai and Henikoff, 2006; L. Comai and E. Buckler, personal communication). The number of genes that can be processed can be adjusted in two dimensions, either screening larger numbers of genes across smaller numbers of mutagenized plants or smaller numbers of genes across larger numbers of plants, as needs and mutation densities dictate. Analysis of sequences for repetitive tracts prior to use in a SequeTILLING experiment. Dot matrix analysis of 18 concatenated genomic sequences totaling 101,174 bp compared against itself. Signals off the diagonal line represent identities that pose potential complicating factors for rapid assembly. Workflow of SequeTILLING. A 48 × 48 grid of individual DNAs is collapsed into 48-row and 48-column pools for gene amplification. Sequencing adapters can be whatever the manufacturer recommends, as long as the 5-bp barcode sequence for each pool is the first part of the sequence read. Sequencing approaches can also apply readily to EcoTILLING, assigning individual barcodes to specific inbred lines or accessions rather than pools of mutants, and then resequencing target genes in these collected lines. Sequencing several independent samples of each accession will robustly distinguish real SNPs from sequencing errors although, because of their inherent variability, these sequences will need to be covered at greater depth to make assignment of SNPs to accessions reliable. One of the important developments to grow out of this quantum leap in throughput is that, even though the research community is still requesting TILLING of a variety of species, the capacity to generate the data is now substantially greater than the user requests for EMS TILLING of specific genes. Consider that a small number of gigabase sequencer runs would quickly equal all the TILLING that has been done to date. The future for these resources may well be that, as mutant populations are being developed and expanded for various grass species, research communities focused on those species need to begin assembling lists of genes for which mutations are desired and develop prioritized approaches for screening them. As massively parallel sequencing methods become more widespread and accessible, the limiting feature of TILLING efforts will be the creation, mutation density testing, curation, and distribution of the mutant populations. Fortunately, once established and especially once shared publicly among researchers, these resources will pay benefits for many years to come as both forward and reverse screening tools. I would like to thank Rita Monde, Dacia Daniel, Courtney Chambers, Leonie Leduc, Heather Sahm, and Tara Breen for technical assistance, and Philip SanMiguel, Paul Parker, Luca Comai, Dick McCombie, and Dick Johnson for valuable discussions.