Bradley J. Till

Discovery of induced point mutations in maize genes by TILLING

BackgroundGoing from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.ResultsWe demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.ConclusionsOur findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.

Discovery of chemically induced mutations in rice by TILLING

BackgroundRice is both a food source for a majority of the worlds population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method.ResultsTo apply TILLING to rice, we developed two mutagenized rice populations. One population was developed by treatment with the chemical mutagen ethyl methanesulphonate (EMS), and the other with a combination of sodium azide plus methyl-nitrosourea (Az-MNU). To find induced mutations, target regions of 0.7–1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes. Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS-treated population and 30 in the Az-MNU population.ConclusionWe estimate that the density of induced mutations is two- to threefold higher than previously reported rice populations (about 1/300 kb). By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project.

TILLING. Traditional Mutagenesis Meets Functional Genomics

Most of the genes of an organism are known from sequence, but most of the phenotypes are obscure. Thus, reverse genetics has become an important goal for many biologists. However, reverse-genetic methodologies are not similarly applicable to all organisms. In the general strategy for reverse genetics that we call TILLING (for Targeting Induced Local Lesions in Genomes), traditional chemical mutagenesis is followed by high-throughput screening for point mutations. TILLING promises to be generally applicable. Furthermore, because TILLING does not involve transgenic modifications, it is attractive not only for functional genomics but also for agricultural applications. Here, we present an overview of the status of TILLING methodology, including Ecotilling, which entails detection of natural variation. We describe public TILLING efforts in Arabidopsis and other organisms, including maize (Zea mays) and zebrafish. We conclude that TILLING, a technology developed in plants, is rapidly being adopted in other systems.

TILLING to detect induced mutations in soybean

BackgroundSoybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the worlds protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).ResultsWe screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (~1/140 kb), while another EMS population had a mutation density of ~1/250 kb. The remaining population had a mutation density of ~1/550 kb. Because of soybeans polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.ConclusionThe development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

A protocol for TILLING and Ecotilling in plants and animals

We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.

High-Throughput TILLING for Functional Genomics

Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

Tilling and Ecotilling for Crop Improvement

The modern crop scientist has a large amount of available nucleotide sequence information to identify genes of potential agronomic importance. Using reverse genetic approaches, specific genes can be disrupted, and hypotheses regarding gene function directly tested in vivo. Although a number of reverse genetic methods have been introduced, many are limited in application because they are organism-specific, expensive, transgenic or only transiently disrupt gene function. However, traditional mutagenesis using chemical mutagens has been widely used as a forward genetics strategy to create many new crop plant varieties at relatively low cost. Mutagens such as ethyl methanesulphonate (EMS), cause stable point mutations and thus produce an allelic series of truncation and missense changes that can provide a range of phenotypes. TILLING (Targeting Induced Local Lesions IN Genomes) uses traditional mutagenesis and SNP discovery methods for a reverse genetic strategy that is high in throughput, low in cost, and applicable to most organisms. Over the past six years, TILLING has moved from proof-of-concept to production with the establishment of publicly available services for Arabidopsis, maize, lotus, and barley. Pilot-scale projects have been completed on several other plant species, including wheat. The protocols developed for TILLING have been adapted for the discovery of natural nucleotide diversity, a method termed EcoTILLING. Like TILLING, EcoTILLING is general and has been applied to plants as diverse as Arabidopsis and poplar. We review here current TILLING and EcoTILLING technologies and discuss the progress that has been made in applying these methods to many different plant species.

Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L)

BackgroundThe TILLING and Ecotilling techniques for the discovery of nucleotide polymorphisms were applied to three potato (Solanum tuberosum) cultivars treated with gamma irradiation. The three mutant cultivars tested were previously shown to exhibit salinity tolerance, an important trait in countries like Syria where increasing soil salinity is affecting agricultural production.FindingsThree gene-specific primer pairs were designed from BAC sequence to amplify ~1 to 1.5 kb of gene target. One of the three primer pairs amplified a single gene target. We used this primer pair to optimize enzymatic mismatch cleavage and fluorescence DNA detection for polymorphism discovery. We identified 15 putative nucleotide polymorphisms per kilobase. Nine discovered polymorphisms were unique to one of the three tetraploid cultivars tested.ConclusionThis work shows the utility of enzymatic mismatch cleavage for TILLING and Ecotilling in different varieties of potato. The method allows for rapid germplasm characterization without the cost and high informatics load of DNA sequencing. It is also suitable for mutation discovery in high-throughput reverse genetic screens.

Induction, rapid fixation and retention of mutations in vegetatively propagated banana

Mutation discovery technologies have enabled the development of reverse genetics for many plant species and allowed sophisticated evaluation of the consequences of mutagenesis. Such methods are relatively straightforward for seed-propagated plants. To develop a platform suitable for vegetatively propagated species, we treated isolated banana shoot apical meristems with the chemical mutagen ethyl methanesulphonate, recovered plantlets and screened for induced mutations. A high density of GC-AT transition mutations were recovered, similar to that reported in seed-propagated polyploids. Through analysis of the inheritance of mutations, we observed that genotypically heterogeneous stem cells resulting from mutagenic treatment are rapidly sorted to fix a single genotype in the meristem. Further, mutant genotypes are stably inherited in subsequent generations. Evaluation of natural nucleotide variation showed the accumulation of potentially deleterious heterozygous alleles, suggesting that mutation induction may uncover recessive traits. This work therefore provides genotypic insights into the fate of totipotent cells after mutagenesis and suggests rapid approaches for mutation-based functional genomics and improvement of vegetatively propagated crops.

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.