Cascade of chromosomal rearrangements caused by a heterogeneous T‐DNA integration supports the double‐stranded break repair model for T‐DNA integration

Summary Transferred DNA (T‐DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double‐stranded break repair (DSBR) pathway is a major model for T‐DNA integration. From this model, we expect that two ends of a T‐DNA molecule would invade into a single DNA double‐stranded break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T‐DNA integration, which has never been observed. In this work, we demonstrated it in an Arabidopsis T‐DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T‐DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T‐DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break‐junction pattern in seb19 is consistent with the result of heterogeneous T‐DNA integration but not of recombination between two T‐DNA insertions. We demonstrated that, in seb19, heterogeneous T‐DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosomes 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T‐DNA integration supports the DSBR model and suggests that two ends of a T‐DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. Significance Statement From the double‐strand break repair (DSBR) model for T‐DNA integration, we expect the occurrence of a heterogeneous T‐DNA integration, in which two ends of a T‐DNA molecule could invade into different DNA double‐strand breaks (DSBs). Here we demonstrate this phenomenon and show how incorrect DNA repairs would lead to chromosomal abnormalities.