In Arabidopsis, one can search for T-DNA insertions in a particular gene through stock centers in the USA, Europe, and Japan with the four most widely used mutant populations being SALK ( Alonso et al., 2003), GABIKat ( Rosso et al., 2003), SAIL ( Sessions et al., 2002), and FLAGdb ( Samson et al., 2002) lines.ĭespite the widespread implementation of T-DNA technology for forward and reverse genetics, there are cases in which causal gene identification can become problematic. The relative ease by which T-DNA insertion sites are identified by PCR-based methods has also enabled the rapid development of reverse genetic resources in plants. The process of insertion site recovery involves primarily polymerase chain reaction (PCR)-based methods, including thermal asymmetric interlaced PCR (TAIL-PCR) ( Liu et al., 1995), inverse PCR ( Ochman et al., 1988), or restriction site PCR ( Ji and Braam, 2010). Because of the known sequences of the inserted T-DNA, recovering the insertion sites in the genome has become a routine operation. In the model plant species Arabidopsis thaliana, the modified T-DNA-based transformation system has been widely used not only to generate loss-of-function mutants via insertional mutagenesis but also for gene overexpression by activation tagging ( Alonso et al., 2003 Rosso et al., 2003 Ichikawa et al., 2006). T-DNA used in mutagenesis is modified from the original tumor-inducing (Ti) plasmid with 25 base pairs direct repeat border sequences at both ends ( Zambryski et al., 1980 Hoekema et al., 1983). The latter method of mutagenesis involves Agrobacterium tumefaciens-mediated transfer-DNA (T-DNA) integration ( Gelvin, 1998). ![]() Forward genetics involves screening a population of plants that has been mutagenized by chemicals, radiation, or biological agents. We present a case study in which TDNAscan was used to determine that the recessive Arabidopsis thaliana hypersensitive to latrunculin B (hlb3) mutant isolated in a forward genetic screen of T-DNA mutagenized plants encodes a class II FORMIN.įorward genetics is an approach used to identify genes that control plant phenotypes of interest. We expect that TDNAscan will be a valuable addition to forward genetics toolkits because it provides a solution to the problem of causal gene identification, particularly genes disrupted by truncated T-DNA insertions. This method, called TDNAscan, was developed to be an open source software. Our method enables the detection of the corresponding T-DNA insertion orientation and zygosity as well as insertion annotation. Here, we present a next generation sequencing (NGS)-based platform to facilitate the identification of complete and truncated T-DNA insertions. Despite the popularity and success of these methods, they have limited capabilities, particularly in situations in which the T-DNA is truncated. Transfer (T)-DNA insertions in mutants isolated from forward genetic screens are typically identified through thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), inverse PCR, or plasmid rescue. Noble Research Institute LLC, Ardmore, OK, United States. ![]() Robinson, Xiaofei Cheng, Jiangqi Wen and Elison B.
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