Transferred DNA (T‐DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T‐DNA‐tagged genomic loci showed that T‐DNA integration resulted in target site deletions of 29–73 bp. In those cases where integrated T‐DNA segments turned out to be smaller than canonical ones, the break‐points of target deletions and T‐DNA insertions overlapped and consisted of 5–7 identical nucleotides. Formation of precise junctions at the right T‐DNA border, and DNA sequence homology between the left termini of T‐DNA segments and break‐points of target deletions were observed in those cases where full‐length canonical T‐DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T‐DNA segments showed no homology to break‐points of target sequence deletions. Homology between short segments within target sites and T‐DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T‐DNA integration results from illegitimate recombination. The data suggest that while the left T‐DNA terminus and both target termini participate in partial pairing and DNA repair, the right T‐DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')H (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')ll enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus-and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.

A recessive pale mutation, designated as cs, was identified by transferred‐DNA (T‐DNA)‐mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T‐DNA, was mapped to the position of the ch‐42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch‐42 mutants indicated allelism. Plant boundaries of the T‐DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full‐length cDNA clones of the wild‐type cs gene. Transformation of the pale mutant with T‐DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T‐DNA induced mutation. DNA sequence comparison of the cs mutant and its wild‐type allele revealed that the T‐DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild‐type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark‐light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

The LmR1 locus, which controls seedling resistance to the blackleg fungus Leptosphaeria maculans in the Brassica napus cultivar Shiralee, was positioned on linkage group N7. Fine genetic mapping in a population of 2500 backcross lines identified three molecular markers that cosegregated with LmR1. Additional linkage mapping in a second population colocalized a seedling resistance gene, ClmR1, from the cultivar Cresor to the same genetic interval on N7 as LmR1. Both genes were located in a region that showed extensive inter-and intragenomic duplications as well as intrachromosomal tandem duplications. The tandem duplications seem to have occurred in the Brassica lineage before the divergence of B. rapa and B. oleracea but after the separation of Brassica and Arabidopsis from a common ancestor. Microsynteny was found between the region on N7 carrying the resistance gene and the end of Arabidopsis chromosome 1, interrupted by a single inversion close to the resistance locus. The collinear region in Arabidopsis was assayed for the presence of possible candidate genes for blackleg resistance. These data provided novel insights into the genomic structure and evolution of plant resistance loci and an evaluation of the candidate gene approach using comparative mapping with a model organism.

Doubled haploid (DH) lines together with a cotyledon bioassay were employed for the molecular analysis of resistance to the blackleg fungus Leptosphaeria maculans in the Australian Brassica napus cultivars Shiralee and Maluka. We used bulked segregant analysis to identify 13 RAPD and two RFLP markers linked to the resistance phenotype and mapped these markers in the segregating DH population. Our data suggest the presence of a single major locus controlling resistance in the cultivar Shiralee, confirming our previous results obtained from Mendelian genetic analyses. In addition, preliminary mapping data for the cultivar Maluka also support a single locus model for resistance and indicate that the resistance genes from 'Shiralee' and 'Maluka' are either linked or possibly identical. The molecular markers identified in this study should be a useful tool for breeding blackleg resistant varieties using marker-assisted selection, and are the essential first step towards the map-based cloning of this resistance gene.