21
TILLING inLotus japonicus
Trevor L. Wang and Fran Robson
Abstract
Following the seminal work on TILLING inArabidopsis thaliana,a
population of EMS-mutagenized plants was established for Lotus
japonicus‘Gifu’to be used for both forward and reverse screening. This
was developed into the Lotus TILLING platform which subsequently
becameRevGenUK covering not only TILLING inL. japonicus, but also
Medicago truncatulaand other species. Over the last 10 years, nearly two
thousand mutations for more than 160 genes have been identified using
the platform. In this article, we cover the history, development and current
technology employed in the process and explore the impact TILLING has
had on Lotus research.21.1 Introduction
Targeting Induced Local Lesions IN Genomes or
TILLING was devised by Claire McCallum and
developed for use with Arabidopsis in Seattle,
USA, under a collaboration between the groups
of Steve Henikoff at the Fred Hutchinson Cancer
Research Centre and Luca Comai at the Uni-
versity of Washington (McCallum et al. 2000 ). It
is a reverse genetics method for identifying
mismatches in DNA heteroduplexes. Initially, the
method relied on denaturing HPLC to identify
the mismatches, but it was later modified to
permit a higher throughput (Colbert et al. 2001 )
and establish the Arabidopsis TILLING platform
(ATP) in Seattle. This latter method uses an
endonuclease, CEL1, from celery and has been
adopted almost universally. It remains the
method of choice, although others have been
used such as high-resolution melting (e.g. Parry
et al. 2009 ). The platform expanded to cover
other species and eventually became the Seattle
TILLING Project (STP), but ceased operation in
2011, with most of the operation being moved to
the Comai lab at UC Davis (http://tilling.ucdavis.
edu/index.php/Main_Page) where it continues to
date.
TILLING is particularly suited to species with
limited genomic information, those that lack
other robust reverse genetic methods and espe-
cially crops, since it is based on established
mutation breeding methods without the use of
genetic manipulation. Furthermore, it generatesT.L. Wang (&)F. Robson
Metabolic Biology, John Innes Centre, Norwich
Research Park, Colney, Norwich NR4 7UH, UK
e-mail: [email protected]
S. Tabata and J. Stougaard (eds.),The Lotus japonicus Genome, Compendium of Plant Genomes,
DOI 10.1007/978-3-662-44270-8_21,©Springer-Verlag Berlin Heidelberg 2014
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