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

Chambers, Kylie, Lowe, Rohan Gt, Howlett, Barbara J, Zander, Manuel, Batley, Jacqueline, Van de Wouw, Angela P and Elliott, Candace 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 . doi:10.1186/s40694-014-0010-y


Author Chambers, Kylie
Lowe, Rohan Gt
Howlett, Barbara J
Zander, Manuel
Batley, Jacqueline
Van de Wouw, Angela P
Elliott, Candace E
Title Next-generation genome sequencing can be used to rapidly characterise sequences flanking T-DNA insertions in random insertional mutants of Leptosphaeria maculans.
Journal name Fungal biology and biotechnology
ISSN 2054-3085
Publication date 2014-12-07
Year available 2014
Sub-type Article (original research)
DOI 10.1186/s40694-014-0010-y
Open Access Status DOI
Volume 1
Total pages 4
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Abstract 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.

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 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.

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.
Keyword Filamentous fungi
Next-generation sequencing
T-DNA mutants
TAIL-PCR
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Agriculture and Food Sciences
 
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Created: Wed, 15 Nov 2017, 13:18:49 EST