T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics - PubMed (original) (raw)

T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics

F E Tax et al. Plant Physiol. 2001 Aug.

Abstract

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 >40-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.

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Figures

Figure 1

Figure 1

Structure of T-DNA LB-flanking region from _emb_88 and of the corresponding region from wild type (WT). A, Rescued LB and plant DNA from the _emb_88 mutant. The 1,626-bp _Eco_RI fragment flanking the _emb_88 T-DNA LB consisted of 80 bp of the extreme T-DNA LB (black), 23 bp of “filler DNA” (white), and 1,523 bp of Arabidopsis genomic sequence (shaded). The genomic region contained the 3′-terminal 484 bp of a gene encoding a novel Arabidopsis Leu-rich repeat (LRR) protein (designated by arrow), and 1,039 bp of single-copy downstream extragenic sequence. B, Diagram of the corresponding genomic region from WT: a 2.3-kbp _Eco_RI fragment containing the prospective site of T-DNA insertion and the regions used as probes for the Southern blots shown in Figure 2. Probe A corresponded to the LB flanking region and probe B to the region predicted to be immediately adjacent to the T-DNA RB.

Figure 2

Figure 2

Southern-blot analysis of genomic DNA from _emb_88 heterozygotes and WT. A, LB-flanking probe detected a T-DNA-associated polymorphism in _emb_88 heterozygotes. Genomic DNA isolated from _emb_88 heterozygotes (H) or WT Arabidopsis (WT) was digested with _Eco_RI or HinDIII, resolved by agarose gel electrophoresis, Southern blotted, and probed with the 1.6-kbp _Eco_RI fragment rescued from the T-DNA LB of the _emb_88 mutant. Arrows indicate positions of bands corresponding to the disrupted allele, in lanes containing DNA from heterozygotes. B, Sequences on the prospective T-DNA RB were not disrupted in _emb_88 heterozygotes. A genomic Southern blot identical to that shown in A was probed with a 250-bp genomic fragment that lies on the “right” side of the prospective T-DNA insertion site, immediately adjacent to the rescued LB sequence in WT DNA (“probe B,” in Fig. 1B). This blot was probed at medium stringency in an effort to detect a disrupted allele in heterozygotes (see “Materials and Methods”). High-resolution scans of autoradiographs are shown.

Figure 3

Figure 3

LRRPK gene structure and identification of a T-DNA insert adjacent to LRRPK sequences. A, Diagram showing the structure of WT LRRPK gene (Deeken and Kaldenhoff, 1997). Positions of PCR primers used in subsequent experiments are shown. B, Map of the PCR fragment obtained from the prospective LRRPK knockout line, _LRRPK_-TKO. The 1,122-bp product contained 102 bp of the T-DNA LB (black), adjacent to 1,020 bp of the LRRPK gene, containing 5′-untranslated region (UTR; 458 bp) and upstream extragenic sequences.

Figure 4

Figure 4

PCR detection of the WT LRRPK allele in _LRRP_K/T-DNA homozygotes. Gene-specific and T-DNA-specific primers were used to detect the presence of the WT and mutant LRRPK alleles in genomic DNA from a representative _LRRPK_-TKO homozygote, and from WT controls. Genomic DNA template (DNA) and primer combinations (Primers) used in each reaction are labeled above each lane. Primers: F, LRRPK-specific forward (5′) primer; R2, LRRPK-specific reverse (3′) primer; LB, T-DNA specific LB primer. Primer positions are indicated on LRRPK gene diagrams in Figure 3. Lanes, in order shown: 1, control amplification of the LRRPK/T-DNA junction product from a representative _LRRPK_-TKO homozygote; 2, control amplification of the WT LRRPK gene from WT genomic DNA; 3, negative control, showing lack of the LRRPK/T-DNA junction product in WT DNA; 4, amplification of the WT LRRPK PCR product from the LRRPK/T-DNA homozygote.

Figure 5

Figure 5

General model for T-DNA-associated rearrangements in _emb_88 and _LRRPK_-TKO. In each case, T-DNA (white) has inserted into a target locus (black) along with LB-flanking sequences originating from a different chromosome (shown in gray). Sequences from this latter chromosome are not associated with the T-DNA RB. e, Presumed location of the gene responsible for the embryo-defective phenotype in the _emb_88 line. Sizes of the translocated LB-flanking regions are ≥than 40 kbp for _emb_88, and at least 1,020 bp for the _LRRPK_-TKO line.

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