Deletion-Based Reverse Genetics in Medicago truncatula      

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.

Translational Genomics in Agriculture: Some Examples in Grain Legumes

“…However, systematic mutant populations have been developed in several species in order to address this issue. TILLING populations are available for L. japonicus, pea, M. truncatula and chickpea (Dalmais et al, 2008;Le-Signor et al, 2009;Perry et al, 2003;Varshney et al, unpublished), Fast Neutron generated deletion mutant populations for M. truncatula, pea and soybean (Bolon et al, 2011;Hofer et al, 2009;Rogers et al, 2009;Smykal et al, 2012) and insertion mutant populations generated using exogenous (Medicago, d 'Erfurth et al, 2003) and endogeneous retrotransposons (Lotus, Urbański et al, 2012) have been generated and have contributed to gene discovery in these species (see Domoney et al, 2013;Domonkos et al, 2013;Urbański et al, 2013).…”

Functional genomics to study stress responses in crop legumes: progress and prospects

“…For example, in the model legumes, Medicago (12 000 M2 plants) (Rogers et al 2009) and Lotus (4904 M2 lines) (Perry et al 2009) mutant populations were developed for use in reverse genetics. In the case of crop legumes, over 3000 M3 lines were developed in common bean and evaluated with root nodulation tests by Porch et al (2009).…”

Mutagenesis in Plant Breeding for Disease and Pest Resistance

“…They induce broad range of deletions and chromosomal changes and are often accompanied by gamma radiation. The major fast neutron bombardment technique is Delete-a-gene, a knockouts gene system for plants Rogers et al, 2009). Delete-a-gene combines fast neutron radiation of seeds and identification of mutants by PCR using two specific primers for targeted locus and shortened PCR extension time to suppress the amplification of a wild type gene.…”