element, a single clonally amplified founder plant
is sufficient to generate a mutant population of
any desired size. Another advantage ofLORE1a
is the absence of untagged phenotypic mutants
derived from somaclonal mutations induced dur-
ing tissue culture. This is often problematic when
TE activation in cell culture is used for con-
struction of mutant collections. SinceLORE1is
endogenous toLotus, theLORE1mutants are not
transgenic, and no biological containment pre-
cautions are required, enabling applications such
as large-scale mutant screening in openfields.
As a pilot experiment, two groups established
medium-scale mutant populations usingLORE1
(Fukai et al. 2012 ; Urbanski et al. 2012 ). From
the two mutant populations composed of 2,450
and 3,744 plant lines, 4,532 and 8,935 insertions
sites were identified, respectively (Fukai et al.
2012 ; Urbanski et al. 2012 ). The large-scale
identification of insertion sites revealed that
LORE1has a preference for insertion into genes,
limiting the number of insertions required for
saturation mutagenesis (Urbanski et al. 2012 ).
20.6 Identification ofLORE1
Insertions in the Mutant
Population by Deep
Sequencing
Mutated genes can be identified by the sequenc-
ing offlanking regions of newLORE1insertions.
A sequence-specific amplified polymorphism
(SSAP) technique has been used for small-scale
experiments of insertion site identification
(Madsen et al. 2005 ; Yokota et al. 2009 ), and a
simplified SSAP protocol has been set up spe-
cifically for identifyingLORE1insertions (Ur-
banski et al. 2013 ). The SSAP method, however,
is not well suited for high-throughput insertion
site identification. Instead, population-wide
characterization of insertion sites was carried out
by taking advantage of molecular barcoding
combined with next-generation sequencing
technology using the FSTpoolit protocol and
software. The laboratory protocol relies on spe-
cific amplification ofLORE1flanking regions
from plants pooled in rows and columns of a two-
dimensional array. Nested Splinkerette PCR,
where molecular barcodes are introduced as part
of the PCR primers, is used to ensure highly
specific amplification of the LORE1 flanking
sequences (Urbanski et al. 2012 , 2013 ). The two-
dimensional setup and barcoding allow pooling
of more than 9,000 plants per sequencing library
and subsequent automated identification of
insertions in all pooled individuals using the
FSTpoolit software package (Urbanski et al.
2012 ).20.7 Line Availability and Current
Status of theLORE1Mutant
PanelFollowing the successful pilot experiments, the
groups in both Japan and Denmark have con-
tinued to expand theLORE1populations. At the
time of writing, theLORE1mutant collection
holds more than 80,000 lines with in excess of
340,000 annotated insertions. The number of
lines is planned to increase to*120,000 during
2014/2015, which will result in more than
450,000 annotated insertions, bringing the
mutant panel to near-saturation with mutants
readily available for the majority ofLotusgenes.
The number of insertions per line ranges from 1
to 15 with an average of four, and all insertions
are annotated with information about gene model
overlaps and details on the number of reads
supporting the insertion call.
AllLORE1lines, including supporting infor-
mation, can be browsed and queried using the
BLAST tools and genome browser at the Kazusa
DNA Research Institute Web site (http://www.
kazusa.or.jp/lotus/index.html) or at the Centre for
Carbohydrate Signalling and Recognition
(CARB) (http://carb.au.dk/lore1/). The Japanese224 E. Fukai et al.