BACKGROUND:Banks of mutants with random insertions of T-DNA from Agrobacterium tumefaciens are often used in forward genetics approaches to identify phenotypes of interest. Upon identification of mutants of interest, the flanking sequences of the inserted T-DNA must be identified so that the mutated gene can be characterised. However, for many fungi, this task is not trivial as widely used PCR-based methods such as thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) are not successful.FINDINGS:Next-generation Illumina sequencing was used to locate T-DNA insertion sites in four mutants of Leptosphaeria maculans, a fungal plant pathogen. Sequence reads of up to 150bp and coverage ranging from 6 to 24 times, were sufficient for identification of insertion sites in all mutants. All T-DNA border sequences were truncated to different extents. Additionally, next-generation sequencing revealed chromosomal rearrangements associated with the insertion in one of the mutants.CONCLUSIONS:Next-generation sequencing is a cost-effective and rapid method of identifying sites of T-DNA insertions, and associated genomic rearrangements in Leptosphaeria maculans and potentially in other fungal species.
CITATION STYLE
Chambers, K., Lowe, R. G., Howlett, B. J., Zander, M., Batley, J., Van de Wouw, A. P., & Elliott, C. E. (2014). Next-generation genome sequencing can be used to rapidly characterise sequences flanking T-DNA insertions in random insertional mutants of Leptosphaeria maculans. Fungal Biology and Biotechnology, 1(1). https://doi.org/10.1186/s40694-014-0010-y
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