Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals

Abstract

With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating / pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.

Keywords: EcoTILLING (Ecotype TILLING); Reverse genetics; SNP (single nucleotide polymorphism); TILLING (target induced local lesions in genomes); functional genomics; genetic stocks.; sequencing.

Fig. (1)

Diagram of the TILLING method in which seeds are mutagenized with a chemical mutagen and germinated to produce M₁ plants. M₁ plants are selfed to produce the M₂ from which DNA is extracted for analysis. The M₂ is allowed to produce seed which can be easily stored for future analysis. Once the DNA is extracted from the mutant population, the DNA is normalized and pooled together. The number of individuals in a pool depends on the ploidy level of the plant and the amount of naturally occurring SNPs, which may require the number of individuals in the pool to be reduced. The targeted gene is amplified using a forward primer with 700 nm dye label and a reverse primer with an 800 nm dye label attached to the 5’ ends. The PCR products are heated and cooled to form heteroduplexes between the accessions in the pool. The resulting pool will contain a mixture of homoduplexes and heteroduplexes. Any mismatches (SNPs or small INDELS) will be detected by a mismatch endonuclease (CEL I) and cleaved into two separate products, which will be detected in the 700 and 800 dye channel of a LI-COR DNA Analyzer. The additive size of the cleaved fragments should equal the total length of the entire product. Once the cleaved fragments and their respective polymorphic site are identified, these individuals are sequenced to verify the induced mutation. EcoTILLING is performed in the same manner except that the seed are not mutagenized; therefore, the process begins by extracting DNA from a reference plant and members of the population and continuing with the remaining steps to determine natural polymorphisms.

Fig. (2)

EcoTILLING images produced from a collection of Vigna radiata. Polymorphic sites are marked with boxes and were subsequently confirmed by sequencing. A size standard (50-700 bp) was included to estimate the size of the cleaved fragments and the target gene.