Abstract
The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. T-DNA and transposon-based mutagenesis has been widely employed but is limited to species in which transformation and tissue culture are efficient. In other species, TILLING (for Targeting Induced Local Lesions IN Genomes), based on chemical mutagenesis, has provided an efficient method for the identification of single base pair mutations, only 5% of which will be null mutations. Furthermore, the efficiency of inducing point mutations, like insertion-based mutations, is dependent on target size. Here, we describe an alternative reverse genetic strategy based on physically induced genomic deletions that, independent of target size, exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron mutagenesis and a sensitive polymerase chain reaction-based detection. A population of 156,000 Medicago truncatula plants has been structured as 13 towers each representing 12,000 M2 plants. The De-TILLING strategy allows a single tower to be screened using just four polymerase chain reaction reactions. Dual screening and three-dimensional pooling allows efficient location of mutants from within the towers. With this method, we have demonstrated the detection of mutants from this population at a rate of 29% using five targets per gene. This De-TILLING reverse genetic strategy is independent of tissue culture and efficient plant transformation and therefore applicable to any plant species. De-TILLING mutants offer advantages for crop improvement as they possess relatively few background mutations and no exogenous DNA.
Highlights
The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants
An indication of the size range of deletions induced by fast neutron bombardment has been made through a review of the mutants catalogued by the National Science Foundation Arabidopsis Information Resource (TAIR; www. arabidopsis.org)
Genomic DNA of dmi1-4 and wild-type M. truncatula A17 were combined in ratios up to 1 mutant in 24,000 wild types to create a set of PCR templates useful for assessing the sensitivity of detection strategies (Fig. 1)
Summary
The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. Systematic reverse genetic platforms, allowing researchers to obtain plants mutated at any identified locus, have streamlined functional genomics in well-resourced model species These platforms are generally based on insertional mutagenesis using T-DNA (Azpiroz-Leehan and Feldmann, 1997; Krysan et al, 1999; Sussman et al, 2000; Sessions et al, 2002; Alonso et al, 2003) or transposon tagging (Martienssen, 1998; Parinov et al, 1999; Speulman et al, 1999; Parinov and Sundaresan, 2000; Tadege et al, 2008), which, through the introduction of known sequences, greatly facilitates gene cloning and allows the high-throughput characterization of insertion sites.
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