T-DNA-Associated Duplication/Translocations in Arabidopsis. Implications for Mutant Analysis and Functional Genomics

Abstract

(D.M.V.) T-DNA insertion mutants have become a valuable resource for studies of gene function in Arabidopsis. In the course of both forward and reverse genetic projects, we have identified novel interchromosomal rearrangements in two Arabidopsis T-DNA insertion lines. Both rearrangements were unilateral translocations associated with the left borders of T-DNA inserts that exhibited normal Mendelian segregation. In one study, we characterized the embryo-defective88 mutation. Although emb88 had been mapped to chromosome I, molecular analysis of DNA adjacent to the T-DNA left border revealed sequence from chromosome V. Simple sequence length polymorphism mapping of the T-DNA insertion demonstrated that a Ͼ40-kbp region of chromosome V had inserted with the T-DNA into the emb88 locus on chromosome I. A similar scenario was observed with a prospective T-DNA knockout allele of the LIGHT-REGULATED RECEPTOR PROTEIN KINASE (LRRPK) gene. Whereas wild-type LRRPK is on lower chromosome IV, mapping of the T-DNA localized the disrupted LRRPK allele to chromosome V. In both these cases, the sequence of a single T-DNA-flanking region did not provide an accurate picture of DNA disruption because flanking sequences had duplicated and inserted, with the T-DNA, into other chromosomal locations. Our results indicate that T-DNA insertion lines-even those that exhibit straightforward genetic behavior-may contain an unexpectedly high frequency of rearrangements. Such duplication/translocations can interfere with reverse genetic analyses and provide misleading information about the molecular basis of mutant phenotypes. Simple mapping and polymerase chain reaction methods for detecting such rearrangements should be included as a standard step in T-DNA mutant analysis.

Approaches to functional genomics in filamentous fungi

“…In plants and fungi, AMT has been shown to cause mutations that are unlinked to the site of T-DNA integration [70,78,79]. The majority of AMT transformants may contain small (~100 bp) genomic deletions [79].…”

“…Even with single-copy T-DNA integrations, small or large genomic rearrangements are frequently observed. These include major chromosomal translocations in a significant proportion of tagged mutants [78,79]. For this reason it is important that putatively T-DNA tagged mutants be tested to see if the T-DNA insertion site is linked to the mutant phenotype.…”

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

“…An embryo-defective88 mutation was characterized with simple T-DNA inserts that exhibited normal Mendelian segregation and detected through a combination of detailed molecular analysis and mapping of the kanamycin-resistance phenotype imparted by the T-DNA (Tax and Vernon, 2001). Although emb88is located on chromosome 1, molecular analysis of genomic DNA directly adjacent to the T-DNA left border revealed sequence from chromosome 5.…”

“…A variety of problems are encountered while analyzing the T-DNA tagged lines, in particular while attempts are made to clone flanking sequences from a T-DNA tagged mutant. Difficulty can arise because of multiple insertions, complex arrangement of T-DNA, insertion of vector backbone sequences, chromosomal duplication and rearrangements or a combination of these (Jorgensen et al 1987, Veluthambi et al 1988, Tax and Vernon, 2001.…”

Disruption ofptLPD1orptLPD2, Genes That Encode Isoforms of the Plastidial Lipoamide Dehydrogenase, Confers Arsenate Hypersensitivity in Arabidopsis

“…The possibility that the T-DNA insertion caused a chromosomal rearrangement (Nacry et al, 1998;Laufs et al, 1999;Tax and Vernon, 2001) was tested by TAIL-PCR. The primers used, T1 and T2, were specific for sequences in the At3g17250/At3g17260 intergenic region immediately adjacent to the putative right border of the T-DNA and pointing toward the insertion site (Fig.…”