Reverse Genetic Strategies in Tomato

The increasing abundance of gene sequence information in tomato and other plants is giving rise to new opportunities for studies of gene function. In a traditional forward genetic approach, a phenotype is investigated to determine the underlying genetic basis of atrait. Inareversegenetic approach, theaimistoalter a gene or its expression level in order to study the resulting phenotype. Ideally, a reverse genetic approach allows a genome wide interrogation of genes, so that the function of every gene can be studied. In tomato, a number of reverse genetic approaches are being developed to leverage the expanding genomic resources in this crop. Among these approaches are insertional mutagenesis, RNA interference (RNAi) and Targeting Induced Local Lesions IN Genomes (TILLING).

In insertional mutagenesis, a collection of lines is generated in which a transposon or modified element has been introduced and has randomly inserted into the genome in order to knock-out or tag genes. This type of approach has been extremely effective in Arabidopsis yielding access to a huge portion of that genome for functional analysis (Alonso et al. 2003). Because Arabidopsis is easily transformed, has a short generation time and has a small genome, large collections of insertion lines are available for this model dicot. In addition, access to the fullysequenced Arabidopsis genome allows mapping of the insertion sites to provide an in silico resource for investigators (Krysanet al. 2002; Sessions et al. 2002). Tomato is less amenable to the development of large populations of insertion lines because transformation methods are less efficient and the tomato genome is much larger than that of Arabidopsis (see Sect. 1.20). Despite these restrictions, a limited number of insertion lines have

2 TILLING is a registered trademark of Arcadia Biosciences been produced and used for reverse genetic studies of gene function in tomato (Meissner et al. 2000; Gi-doni et al. 2003). In some cases, the insertion element can be remobilized into nearby sequences in order to expand the utility of these collections (Gidoni et al. 2003).

Another useful tool for reverse genetics studies in plants is gene silencing through RNAi (Kusaba 2004; Watson et al. 2005). In this method, a transgene designed to produce a self-complementary RNA that forms a double stranded (ds) structure is introduced into a plant by transformation. The dsRNA is processed to small 21 to 24 nucleotide fragments that are then used to target sequence-specific degradation of endogenous RNA. In this way, expression of a target gene of interest can be suppressed and the resulting phenotype can be evaluated. However, the extent of gene silencing through RNAi can vary, so a number of transformants must be screened to ensure that suppression is achieved. In tomato, RNAi has been effectively applied to reduce the expression of a fruit allergen using transformation (Le et al. 2006). A major advantage of RNAi is that it can be used to simultaneously suppress multiple related genes as well as genes in tightly linked tandem arrays (Kusaba et al. 2003; Lawrence and Pikaard 2003). Another advantage of RNAi is that it can be used for tissue-specific suppression of genes when it would be detrimental to eliminate the expression of a gene throughout the plant. For example, fruit specific suppression of tomato DET1 by RNAi resulted in increased carotenoids and flavonoids in fruit and left the remainder of the plant unaffected (Davuluri et al. 2005). An alternate RNAi approach called virus-induced gene silencing (VIGS) utilizes a target sequence inserted into a viral genome to transiently infect plants and suppress target gene expression. In tomato, VIGS has been used to suppress genes expressed during fruit development (Fu et al. 2005). In addition, a high-throughput system for VIGS called Agrodrench has been developed to suppress expression of target genes in roots of tomato and other Solanaceae (Ryu et al. 2004).

TILLING is a powerful reverse genetic technique that allows a researcher to identify genetic variation in a gene of interest (Colbert etal. 2001; McCallum et al. 2000a). TILLING, much like a forward genetic screen, begins with a population or library of individuals with increased levels of genetic variation induced through mutagenesis. However, unlike forward genetic screens, TILLING ends in the identification of genetic lesions at the molecular level, which can then be studied at the phenotypic level. Specifically, a mutagen is used to induce genetic variation in a population of thousands of individuals, from which ge-nomic DNA and seed libraries are generated. These libraries serve as long-lasting, renewable repositories for the selection of plants whose genomes contain mutations of interest. The DNA library is pooled to increase efficiency and screened over targeted regions ofthegenometodiscovernovelgeneticvariation. Mutations at the level of SNPs can be identified rapidly and economically through enzymatic cleavage of mismatched DNA using CEL I endonuclease followed by gel electrophoresis of the fragments. The number of mutations that are discovered depends upon the size of the library, its mutation frequency, and the size of the target gene to be screened. These new mutations form an allelic series with a range of potential phenotypic effects that can help assess gene function (Hirschi 2003).

Reverse genetic approaches have proven extremely useful to study basic gene function. They also offer the potential to speed the development of novel commercial cultivars since functional genomics findings associated with traits of interest in one crop may also be relevant to another. TILLING, in particular, provides a means to access almost any gene for functional studies by increasing the genetic diversity of domestic tomato lines and is also suitable as a non-transgenic means of crop improvement.

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