Quantitative and structural analyses of T-DNA tandem repeats in transgenic Arabidopsis SK mutant lines

Complex T-DNA repeat structures are a frequent outcome of Agrobacterium-mediated plant transformation and often lead to co-suppression of gene expression, even gene silencing. Thus, they are undesired in transgenic plants for commercial or research purposes and frequently need to be efficiently identified in a large transgenic plant population. Application of conventional Southern blot analysis is limited because it is laborious and time consuming. In this study, a new advancement that enables for high throughput determination of T-DNA repeat copy numbers in large scale screening of transgenic plant populations was developed by improving on the standard addition quantitative PCR method with specific reference plasmids. The plasmids contained the Arabidopsis single copy gene encoding high mobility group A and either a T-DNA direct repeat or T-DNA BAR gene. The improvement measured complex T-DNA repeat numbers quickly and accurately. Discrepancies in qPCR detected T-DNA copy number versus Southern detected T-DNA insertion number were largely a result of complex T-DNA repeats. Sequencing results revealed the existence in the repeat junctions of similar T-DNA insertion patterns commonly found in transgenic plants, including precise RB structure and LB deletion. Moreover, the end-joining nucleotides from both RB and LB in many repeat junctions were exactly at sites immediately adjacent to a homologous segment in the two borders, suggesting possible involvement of homologous recombination in the repeat formation. Our study demonstrates that this new advancement in high throughput quantification of T-DNA tandem repeats is useful for a large scale transgenic plant population analysis and in elucidating the mechanism of T-DNA tandem repeat formation.