Revealing the complexities of genetically modified plant genomes
Following their successful sequencing and assembly of a highly contiguous and accurate Arabidopsis thaliana genome in just 4 days using a single MinION flowcell1, the team at the J. Craig Venter Institute, together with researchers from the Salk Institute, turned their attention to using long-read nanopore sequencing to characterise transformed, genetically modified lines2,3.
Genetically modified (GM) plants have been in existence since 1982, when researchers developed a tobacco plant that expressed bacterial antibiotic resistance genes4. Since this time, GM crops have been produced with a range of beneficial traits, including increased yield, herbicide and disease resistance, and enhanced product characteristics such as longer shelf life.
One of the most common methods for introducing new genetic material into plant cells is through the use of the bacterium Agrobacterium tumefaciens. This plant pathogen randomly inserts DNA contained within its plasmid to double strand breaks within the host genome. For both scientific and regulatory reasons, it is important to characterise the insertion sites and copy number of this transfer DNA (T-DNA); however, traditional analysis techniques, such as Southern blotting, can be laborious and lack resolution.
To address this challenge, the research team utilised both optical mapping and long-read nanopore sequencing to examine the genome of two transformed and one reference line of A. thaliana — with each line being fully sequenced on single MinION flow cells. Highly contiguous genomes were assembled with complete chromosome arms being contained within just one or two contigs. Genome analysis allowed the team to resolve T-DNA structures up to 36 kb in length and revealed large-scale T-DNA associated translocations and exchange of chromosome arm ends.
Moreover, sequence contigs for the two transgenic lines (SAIL_232 and SALK_059379) captured up to 39 kb of assembled T-DNA insertion sequence, providing sufficient information to better understand the complexity of such Agrobacterium-mediated transgene insertions (e.g. rearrangements, insertions, deletions, etc.) and the effect of these insertions on proximal genes.
Nanopore sequencing allowed the resolution of T-DNA structures up to 36 kb in length3.
This study provides new insights into the structural impact of engineering plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes. The team now plans to utilise the nanopore sequencing data to identify the methylation status of their transformed genomes, with a view to the potential replacement of separate bisulfite sequencing-based methylation analysis. Commenting on this research, Professor Todd Michael remarked:
‘It has been known that [T-DNA] insertions have variable length and that there are many of them but, up until Oxford Nanopore technology with long reads, it was really impossible see what those insertions looked like’2.
This case study is taken from the plant white paper.
- Michael, T.P. et al. High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell. Nat Commun. 9(1):541 (2018).
- Michael, T. The complex architecture of plant T-DNA transgene insertions. Presentation. Available at: https://nanoporetech.com/ resource-centre/complex-architecture-plant-t-dna-transgene-insertions [Accessed: 10 July 2018]
- Jupe, F. et al. The complex architecture of plant transgene insertions. bioRxiv 282772 (2018).
- Fraley, R.T. et al. Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. U.S.A. 80 (15): 4803–07 (1983).