Abstract
We previously described the identification of a chromosomal deletion in Arabidopsis thaliana that resulted in the elimination of genomic DNA between two T-DNA insertions located ca. 25 kilobases apart on chromosome IV. The mechanism responsible for this deletion appears to have been recombination between the closely spaced T-DNA elements located in trans in a parent plant. In our original study, we observed one such deletion event after screening ca. 2,000 seedlings using a polymerase chain reaction (PCR) assay. Because a method for precisely deleting a selected region of the Arabidopsis genome would have significant value as a research tool, we were interested in determining the frequency with which this type of T-DNA-directed deletion occurs. To do this we designed a genetic screen that would allow us to phenotypically screen for deletions caused by recombination between T-DNA inserts. This screen involved crossing T-DNA single-mutant lines in order to produce F1 plants in which the two T-DNA insertions flanked a FUSCA (FUS) locus present in the genome. Loss-of-function mutations of FUS genes cause a distinctive developmental phenotype that can be easily scored visually in young seedlings. We used T-DNA lines flanking FUS2, FUS6, FUS7, and FUS11 for this study. Recombination between the T-DNAs in an F1 parent should result in deletion of the FUS gene located between the T-DNAs. Because the deletion would be heterozygous in the F2 generation, we screened the F3 progeny of pooled F2 individuals to search for the fus loss-of-function phenotype. Using this strategy we were able to evaluate a total of 28,314 meioses for evidence of deletions caused by recombination between the T-DNA inserts. No seedlings displaying the fus phenotype were recovered, suggesting that deletions caused by recombination between T-DNA inserts are relatively rare events and may not be a useful tools for genome engineering in Arabidopsis.
Highlights
In order to understand the function of a genome, it is critical to have tools available for modifying the DNA sequence of that genome
We use the term ‘‘deletion caused by transferred DNA (T-DNA) recombination’’ to refer to the situation where meiotic crossing over between T-DNA insertions located nearby to each other leads to deletion of the intervening genomic DNA (Fig. 1)
Our objective was to screen a larger population of individuals in order to better ascertain the frequency with which deletions caused by T-DNA recombination occur
Summary
In order to understand the function of a genome, it is critical to have tools available for modifying the DNA sequence of that genome. The T-DNA elements are inserted ‘‘randomly’’, the existence of such a large catalog of lines means that most of the genes in Arabidopsis have at least one T-DNA mutant allele available. These public T-DNA collections are an extremely popular community resource for studying gene function in Arabidopsis. Because approximately 17% of the genes in Arabidopsis are members of a tandemly-duplicated gene family, it can be challenging to study gene function using T-DNA knockouts when the gene of interest has a second copy nearby on the same chromosome (Initiative, 2000) If these duplicated genes display functional redundancy, one must knockout both genes in order to reveal a mutant phenotype. Because of the extremely tight genetic linkage between tandemly duplicated genes, it is not practical to rely on meiotic recombination to produce a double-mutant for tandemly duplicated genes using T-DNA insertion lines
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