21.6 The Future
For more than 10 years, TILLING in Lotus has
been the predominant reverse genetic platform
for this species, primarily because relatively few
laboratories had efficient conventional (rather
than hairy root) transformation systems and no
insertional mutagenesis population had been
developed. With the development of the LORE1
transposon population (see Chap. 20), the play-
ingfield has changed and Lotus TILLING will
no doubt take a backstage since most researchers
prefer knockout mutations as an indicator of gene
function, especially in the initial stages of their
research. Since it is the only way of producing a
range of stable allelic variation, however,
TILLING will remain the most effective and
refined way of allowing detailed investigations of
gene function. Furthermore, one cannot uncover
the function of a gene which when fully dis-
rupted is lethal, other than by producing a range
of weaker, non-lethal alleles.
The advent of efficient resequencing technol-
ogies will also have an impact on Lotus TILL-
ING. As the cost of resequencing falls, it will
become cost-effective to identify all the mutations
in our populations, thus making conventional
TILLING redundant in favour of in silico TILL-
ING (Wang et al. 2012 ); this is already in hand for
species with small genomes or species such as
hexaploid wheat. Each wheat plant can carry
large numbers of mutations, and hence, such
species require the resequencing of relatively
small plant populations (thus reducing the cost) to
identify numerous mutations in each gene. The
future for conventional TILLING inLotus japo-
nicus,therefore, will be briefer than its past.
References
Andersen SU, Cvitanich C, Hougaard BK, Roussis A,
Grønlund M, Jensen DB, Frøkjær LA, Jensen EØ
(2003) The glucocorticoid-inducible GVG system
causes severe growth defects in both root and shoot
of the model legumeLotus japonicus. Mol Plant-
Microbe Interact 16:1069– 1076
Asamizu E, Shimoda Y, Kouchi H, Tabata S, Sato S
(2008) A positive regulatory role forLjERF1in the
nodulation process is revealed by systematic analysis
of nodule-associated transcription factors ofLotus
japonicus. Plant Physiol 147:2030– 2040
Bustos-Sanmamed P, Bazin J, Hartmann C, Crespi M,
Lelandais-Brière C (2013) Small RNA pathways and
diversity in model legumes: lessons from genomics.
Front Plant Sci 4, art 236. doi:10.3389/fpls.2013.00236
Credali A, García-Calderón M, Dam S, Perry J, Díaz-
Quintana A, Parniske M, Wang TL, Stougaard J, Vega
JM, Márquez AJ (2013) The K+-dependent asparagi-
nase, NSE1, is crucial for plant growth and seed
production inLotus japonicus. Plant Cell Physiol
54:107– 118
Colbert T, Till BJ, Tompa R, Reynolds S, Steine MN,
Yeung AT, McCallum CM, Comai L, Henikoff S
(2001) High-throughput screening for induced point
mutations. Plant Physiol 126:480– 484
Den Herder G, Yoshida S, Antolín-Llovera M, Ried MK,
Parniske M (2012)Lotus japonicusE3 Ligase SEVEN
IN ABSENTIA4 destabilizes the symbiosis receptor-
like kinase SYMRK and negatively regulates rhizobial
Infection. Plant Cell 24:1691– 1707
Dong Z-C, Zhao Z, Liu C-W, Luo J-H, Yang J, Huang W-
H, Hu X-H, Wang Tl, Luo D (2005) Floral patterning.
InLotus japonicus. Plant Physiol 137:1272– 1282
Gaude N, Tippmann H, Flemetakis E, Katinakis P,
Udvardi M, Dörmann P (2004) The galactolipid
digalactosyldiacylglycerol accumulates in the peribac-
teroid membrane of nitrogen-fixing nodules of soy-
bean and Lotus. J Biol Chem 279:34624– 34630
Groth M, Kosuta S, Haage K, Hardel S, Gutjahr C,
Brachmann A, Sato S, Tabata S, Findlay K, Wang T,
Parniske M (2013) TwoLotus japonicussymbiosis
mutants impaired at distinct steps of arbuscule devel-
opment. Plant J 75:117– 129
Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till
BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE,
Odden AR et al (2003) Spectrum of chemically
induced mutations from a large-scale reverse-genetic
screen in Arabidopsis. Genetics 164:731– 740
Grønlund M, Gustafsen C, Roussis A, Jensen D, Nielsen
L, Marcker K, Jensen E (2003) TheLotus japonicus
NDXgene family is involved in nodule function and
maintenance. Plant Mol Biol 52:303– 316
Guether M, Neuhäuser B, Balestrini R, Dynowski M,
Ludewig U, Bonfante P (2009) A mycorrhizal-specific
ammonium transporter fromLotus japonicusacquires
nitrogen released by arbuscular mycorrhizal fungi.
Plant Physiol 150:73– 83
Hakoyama T, Watanabe H, Tomita J, Yamamoto A, Sato
S, Mori Y, Kouchi H, Suganuma N (2009) Nicotian-
amine synthase specifically expressed in root nodules
ofLotus japonicus. Planta 230:309– 317
Horst I, Welham T, Kelly S, Kaneko T, Sato S, Tabata S,
Parniske M, Wang TL (2007) TILLING mutants of
Lotus japonicusreveal that nitrogen assimilation and
fixation can occur in the absence of nodule-enhanced
sucrose synthase. Plant Physiol 144:806– 820
Imaizumi-Anraku H, Takeda N, Charpentier M, Perry J,
MiwaH,UmeharaY,KouchiH,MurakamiY,MulderL,21 TILLING inLotus japonicus 241