20.3 Legume Mutant Collections
From the very beginning of the history of genetics,
marked by Mendel’s work on garden peas,
legumes have been extensively exploited as
research tools (Reid and Ross 2011 ). They include
many agronomically important food crops such as
soybean, pea, and peanuts as well as pasture crops
including alfalfa (Medicago sativa), clover
(Trifolium repensandTrifolium pratense), and
bird’s foot trefoil (Lotus corniculatus). Symbiotic
nitrogenfixation is a trait of major agronomical
importance for legumes, and much legume
research has been focused on identifying the
genetic components regulating the symbiotic
interaction. Reflecting the importance of legumes
in research and breeding, several mutant collec-
tions are being established in different legume
species (Imaizumi et al. 2005 ; Triques et al. 2007 ;
Tadege et al. 2008 ; Rogers et al. 2009 ; Bolon et al.
2011 ; Hancock et al. 2011 ; Mathieu et al. 2009 ;
Perry et al. 2003 ;Pislariu et al. 2012 ; Cui
et al. 2013 ). One of them is theLORE1insertion
mutant resource, which was generated in L.
japonicususing the endogenous retrotransposon
LORE1. At the time of writing, it was the only non-
transgenic legume insertion mutant collection.
20.4 Identification ofLORE1and Its
Germline Transposition
LORE1elements constitute a family ofGypsy
retrotransposons endogenous to Lotus. They
contain a chromodomain at the C-terminal of the
integrase, which categorizes them as chromovi-
ruses (Gorinsek et al. 2004 ).LORE1wasfirst
identified because of its insertion into genes
required for symbiotic nitrogenfixation (Madsen
et al.2005;Schauser et al. 1999 ). The symbiotic
mutants were isolated from a gene tagging
population established by introducing the
exogenous maizeAc/Ds DNA transposon into
Lotus (Thykjaer et al. 1995 ). In addition to
LORE1, transpositions of anotherGypsyretro-
transposon Lotus Retrotransposon 2(LORE2)
was also identified in the Ac/Ds population. This
suggested that at least two different endogenous
retrotransposon families were concurrently active
in the Ac/Ds population. Their transpositions
were also identified in other plant populations
regenerated from non-transformed cultured cells
(Umehara et al., personal communication; Fukai
et al. 2010 ). The only condition shared by the
two activation events was the tissue culture step,
suggesting that the activation of LORE1 and
LORE2was associated with tissue culture.
Fukai et al. ( 2010 ) found thatLORE1a, one of
theLORE1family members, can be epigeneti-
cally activated in the regenerated intact plants
from dedifferentiated cells of theLotusB-129
(Gifu) accession. They also found that activated
LORE1atransposes in the germ line, mainly in
pollen, but not during tissue culture. In agree-
ment with thisfinding, the promoter ofLORE1a
showed high activity in pollen (Fukai et al.
2010 ). Variation in DNA methylation patterns in
theLORE1apromoter region among regenerated
plants suggested instability of epigenetic regula-
tion ofLORE1aduring tissue culture (Fukai et al.
2010 ), indicating that tissue culture processes
could induce epigenetic activation ofLORE1a.
LORE2 transpositions were also observed in
plants with activeLORE1copies (Fukai et al.
2012 ), suggesting that activation of LORE2
could be induced in a similar way toLORE1.
Although the precise characteristics of the
transpositional pattern of LORE2 remains
unknown, its transposition frequency was an
order of magnitude lower than that seen for
LORE1ain the mutagenized populations (Ur-
banski, personal communication).20.5 Establishment of aLotus
Mutant Collection Using
the Germline-Specific
RetrotransposonLORE1Establishment of mutant collections requires a
large number of independent insertions, and the
germ line-specific transposition of LORE1a
greatly facilitates large-scale mutagenesis. Since
the number of independent insertions increases in
proportion to the number of seeds harvested from
a founder plant carrying an active LORE1a20 Forward and Reverse Genetics... 223