Journal article

Next-generation genome sequencing can be used to rapidly characterise sequences flanking T-DNA insertions in random insertional mutants of Leptosphaeria maculans

Kylie Chambers, Rohan GT Lowe, Barbara J Howlett, Manuel Zander, Jacqueline Batley, Angela P Van de Wouw, Candace E Elliott

Fungal Biology and Biotechnology | BioMed Central | Published : 2014

Abstract

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 150 bp and cover..

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