Update on Tagged and TILLING Populations in Legumes
Mutagenesis and Beyond! Tools for Understanding
Legume Biology1
Million Tadege2, Trevor L. Wang2, Jiangqi Wen, Pascal Ratet, and Kirankumar S. Mysore*
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (M.T., J.W., K.S.M.);
Department of Metabolic Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom (T.L.W.);
and Institut des Sciences du Végétale, Unité Propre de Recherche 2355, CNRS, 91198 Gif sur Yvette,
France (P.R.)
The family Leguminosae is one of the largest families of flowering plants and includes important crop
legumes such as soybean (Glycine max) and lentil (Lens
culinaris) and forage legumes like alfalfa (Medicago
sativa). Legumes vary in habit from annual to perennial and in their genomes from simple diploids to large
and complex polyploids. Two legume species, Medicago
truncatula and Lotus japonicus, are being used as models
to study legume genetics and genomics. Both of these
species belong to the Galegoid clade that includes pea
(Pisum sativum) and alfalfa but is also closely related to
the other agriculturally important major clade, the
Phaseoloid, that includes bean (Phaseolus vulgaris). M.
truncatula and L. japonicus thus represent very useful
model systems to study pertinent issues that are relevant to economically vital legumes. These two species
were chosen as models because of their diploid, relatively small genomes (450–550 Mb), self-fertility,
relative ease of genetic transformation, and short generation time. The success of any species as a molecular
genetic model depends on the availability of versatile
genetic and genomic resources. The genomes of both of
these species are being sequenced by international
consortiums and are approaching completion (Sato
et al., 2008; Young and Udvardi, 2009). In addition to
the excellent transcriptomics, proteomics, metabolomics, and bioinformatics tools being developed for
both species by different groups around the world, a
sizeable number of mutants have been collected using
various mutagenesis approaches. In this update, we
will briefly describe the current mutant populations
and draw particular attention to two of the most
frequently used mutant resources for reverse genetics:
the Tnt1-tagged population of M. truncatula and the
1
This work was supported by the Samuel Roberts Noble Foundation, the National Science Foundation (Plant Genome grant no.
DBI 0703285), the European Union (Grain Legumes Integrated
Project grant no. FOOD–CT–2004–506223), and the Biotechnology
and Biological Sciences Research Council.
2
These authors contributed equally to the article.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Kirankumar S. Mysore ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.109.144097
978
targeting-induced local lesions in genomes (TILLING)
population of L. japonicus.
LEGUME MUTAGENESIS RESOURCES
Mutagenesis is a fundamental approach in biology
to identify gene function, and in plants it may involve
using chemicals, ionizing radiation, or specific DNA
insertion sequences. All of these have been attempted
in legumes including the models M. truncatula and L.
japonicus, and comprehensive tools for M. truncatula
have been described in a recent review (Ane et al.,
2008).
Chemical Mutagenesis
Alkylated agents such as ethylmethane sulfonate
(EMS) have been widely used in the past for producing
mutagenized populations, which can then be used for
forward genetic screens. It is applicable readily to most
plant species, inducing single base pair G/C-to-A/T
substitutions in nucleotides. TILLING (McCallum
et al., 2000; Colbert et al., 2001) is a reverse genetic
tool that is used to identify these single base pair
changes in target DNA sequences and is likewise
readily applicable to most plants (Henikoff and Comai,
2003). It is frequently carried out in parallel with
forward screens, permitting preselection of deleterious
mutations (Perry et al., 2003). TILLING involves the
identification of mismatches in heteroduplexes formed
by single-stranded DNAs from wild-type and mutant
sequences, and key to the technology are an appropriately mutagenized population of M2 plants and a
method for identifying the mismatches. The sequences
are generated by PCR amplification from a population
of DNAs isolated from single M2 plants using labeled
primers appropriate for the type of detection employed. The DNAs can be pooled to increase the
efficiency of the process. They are then heated, causing
strand separation, reannealed to form the heteroduplexes, and cleaved at the mismatch, and the products
are separated by electrophoresis. Cleavage is carried
out using endonucleases, the most commonly employed, CelI, being readily obtained from celery
(Apium graveolens; Till et al., 2003), although several
others have also been examined (Till et al., 2004;
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Tagged and TILLING Populations in Legumes
Triques et al., 2007). Each cleavage product is labeled
with a single dye. Mutations can readily be identified,
since the enzyme only cuts on the 3# side of the
mismatch, liberating single strands of complementary
sizes that add up to the original amplicon size generated by the primer pair, thus reducing the number of
false-positive observations.
Deletion Mutagenesis
Fast-neutron bombardment (FNB) and g-rays result
in deletion of DNA fragments of variable length from
the genome (Li and Zhang, 2002). FNB populations of
M. truncatula var Jemalong A17 are being developed
by the groups of G. Oldroyd at the John Innes Centre
(Rogers and Oldroyd, 2008) and R. Chen at the Samuel
Roberts Noble Foundation (Wang et al., 2006). Approximately 156,000 M2 families at the John Innes
Centre and 460,000 M2 families at the Noble Foundation have been generated so far, and both populations
have been publicly available for forward and reverse
genetic screening. A reverse screening strategy called
deletion TILLING has been developed. It uses a very
sensitive PCR-based protocol to screen deletion mutants and detects mutants at a frequency of 20% to 50%
using a portion of the mutant population. Forward
genetic screens have also been used to identify mutants with phenotypic alterations from the wild type.
Developments are under way to expedite gene cloning
using the NimbleGen tiling array (R. Duvvuru Muni
and R. Chen, personal communication). FNB has also
been used in soybean (Men et al., 2002) and is being
developed as a resource for this legume (http://rhdev.
webapps.uga.edu/staging/soy_nsf/fastneutron/index.
php).
Insertion Mutagenesis
Insertions such as T-DNA, transposons, or retrotransposons that disrupt gene functions have been
used in both model legumes. T-DNA tagging was
successfully tested in M. truncatula (Scholte et al.,
2002), but so far only about 1,000 lines have been
generated, and screening the population for symbiotic
and morphological mutants suggests that the rate of
somaclonal variation is very high (Brocard et al., 2006).
T-DNA has also been used in L. japonicus (Schauser
et al., 1998), but again to a limited extent. The maize
(Zea mays) transposon Ac/Ds has been used to isolate
important genes in L. japonicus such as the nodulation
transcription factor NIN (Schauser et al., 1999). In
soybean, there has been a recent initiative to develop a
sizeable number of mutants using the maize Ds element (Mathieu et al., 2009). In M. truncatula, however,
the maize En/Spm and the Arabidopsis (Arabidopsis
thaliana) Tag1 transpose poorly (d’Erfurth et al., 2003b,
2006); thus, large-scale tagged populations, other than
Tnt1-tagged lines, are not available.
The transposable element of tobacco (Nicotiana tabacum) cell type 1 (Tnt1) is a very active 5.3-kb copia-type
retrotransposon isolated from tobacco cell cultures
(Grandbastien et al., 1989). Retrotransposons are attractive insertional mutagens for large genomes, since
they result in multiple insertions per genome in one
generation. Unlike the classical DNA transposons (e.g.
Ac/Ds), which excise and reinsert during transposition, long terminal repeat retrotransposons replicate
during transposition and only the new copies move
and insert into new locations (Kumar and Bennetzen,
1999). During this process, the retroelement is first
transcribed into an mRNA, which is then reverse
transcribed by self-encoded reverse transcriptase to
make new retroelements that reinsert in various locations in the genome. Although retrotransposons constitute the major proportion of large and complex
plant genomes, and the number of active retrotransposons is steadily rising, Tnt1 and the rice (Oryza
sativa) Tos17 are the only two long terminal repeat
retroelements that have been used extensively for
large-scale insertional mutagenesis in higher plants
(Miyao et al., 2003; Tadege et al., 2005; Tadege and
Mysore, 2006). Active endogenous retrotransposons
have also been identified in both L. japonicus and M.
truncatula. LORE1 and LORE2 in L. japonicus are Ty3/
gypsy-like retroelements (Fukai et al., 2008) that have
similar potential for isolating knockout mutants in this
model to the use of Tnt1 in M. truncatula. Likewise,
MERE1 in M. truncatula is a copia-like element found
to transpose during tissue culture at a reduced frequency when compared to the heterologous Tnt1
(Rakocevic et al., 2009).
The advantages of Tnt1 as an insertional mutagen
in M. truncatula are numerous: insertions are stable
during seed-to-seed generation in greenhouses; transposition can be activated by tissue culture; new transpositions show preference toward transcribed regions
without a strong preference for a unique target site
sequence (d’Erfurth et al., 2003a; Le et al., 2007; Tadege
et al., 2008); it inserts always as a single copy at one
locus, generating only a 5-bp duplication (target-site
duplication), with none of the deletion or local
genomic rearrangements observed with the T-DNA.
These latter characteristics are important features for
the identification of the tagged loci. Tnt1, therefore,
can target any part of the genome, but it does so more
frequently in the gene-dense regions than the centromeric and other repetitive sequence regions. The preference for exons is a valuable attribute for efficient
saturation mutagenesis (as the M. truncatula genes are
not uniformly distributed in the genome) and may
simply be due to a bias for GC-rich against AT-rich
sequences (Tadege et al., 2008).
M. TRUNCATULA TNT1-TAGGED RESOURCES
The Tnt1 retroelement was initially introduced into
M. truncatula and shown to transpose in tissue culture
by the group of P. Ratet (d’Erfurth et al., 2003a). The
group of K. Mysore at the Noble Foundation then
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Tadege et al.
collaborated with Dr. Ratet’s group and launched the
first large-scale Tnt1 tagging in the R108 background
at the end of 2003. Since then, a multinational European group consisting of 10 laboratories, organized in
the framework of the European Union Grain Legumes
Integrated Project (GLIP), has successfully generated
6,000 Tnt1 lines in the M. truncatula Jemalong 2HA
background (www.eugrainlegume.org). An Italian
group has also recently reported the generation of
1,000 Tnt1 lines in the R108 background (Porceddu
et al., 2008). To date, approximately 15,000 independent Tnt1 lines have been generated in the United
States, and the average copy number per genome
estimated by Southern-blot analysis and sequencing of
flanking sequence tags (FSTs) from a limited number
of lines is approximately 25 (Tadege et al., 2008),
suggesting that the existing population contains approximately 375,000 Tnt1 inserts. Assuming the M.
truncatula genome size to be 500 Mb and the average
gene length to be 1.7 kb, it was previously estimated
that 14,000 Tnt1 lines would provide approximately
90% probability of tagging any average-sized singlecopy gene. To date, 90% of the genes that have been
targeted were found to be tagged in a subpopulation of
10,000 lines, confirming this estimate. This suggests
that the Tnt1 population should provide a very high
probability of tagging any genes in M. truncatula
except very small genes, which unfortunately are not
uncommon in the M. truncatula genome. This is a very
efficient resource that the legume community is encouraged to make the most use of it. The vast array of
Tnt1-tagged genes already identified by FST sequencing that have been described in other species can be
searched at http://bioinfo4.noble.org/mutant/database.
php. Other Tnt1-tagged genes that are identified by
forward and reverse screening include symbiotic
genes (such as NIN, NSP1/2, ERN1, DMI1/3, NFP,
and SUNN), flowering time and floral development
genes, leaf and inflorescence development genes, and
several transcription factors that belong to homeobox,
MADS box, MYB, and zinc finger classes as well as
several metabolic enzymes and various transporters,
which are the subject of active investigation by a large
number of groups.
The Tnt1 population at the Noble Foundation is
generated from a single parental line, Tnk88-7-7, that
contains five copies of the Tnt1 element. Tnk88-7-7 was
obtained by introducing the Tnt1-containing construct
into the R108 genotype and subsequent selfing. This
parental line contains only transposed Tnt1 copies, not
the original construct. Extensive PCR analysis using
sequence-specific primers and sequence analysis of
thermal asymmetric interlaced (TAIL)-PCR fragments
found no antibiotic resistance markers, left and right
T-DNA borders, or any other sequences of the transformation vector. The vector was detected in the
progenitor of Tnk88-7-7 but not in Tnk88-7-7 itself,
suggesting that the Tnt1 element had transposed during the transformation process. The vector and the
transposed Tnt1 copies ended up in different plants by
segregation during the selfing process in soil. The
pipeline for generation of the population is schematically depicted in Figure 1 and has three components
for community utilization: forward genetic screening
of mutant phenotypes; reverse genetic screening of
DNA pools; and a FST database. In brief, mutagenesis
is accomplished by cultivating leaf explants from the
parental line on auxin- and cytokinin-containing culture media and regenerating new plants via somatic
embryogenesis (Tadege et al., 2005, 2008). Each new
plant is derived from a single leaf explant and represents a truly independent line.
At the Noble Foundation, an annual open and free
Tnt1 screening workshop have been organized to
catalogue mutant phenotypes and make them available to the scientific community. The first such screening was conducted in the summer of 2005 involving
various groups from France, the United Kingdom, and
the United States primarily interested in nodulation
biology. To date, approximately 7,000 lines have been
screened for phenotypes ranging from symbiotic and
root development to leaf architecture and shoot meristem functions. Some representative root and nodule
phenotypes are shown in Figure 2. Tnt1 tagging pro-
Figure 1. M. truncatula Tnt1 resource development pipeline. Tnt1
mutagenesis starts with a single parental line (Tnk88-7–7) that contains
five Tnt1 copies that were initially introduced into the R108 background by transformation. Leaf explants from the parental line are
cultured on medium containing auxin and cytokinin to form calli. Tnt1
copies move to new locations and mutagenize the genome during the
first few weeks of the callus formation process. New plants are
regenerated from the calli via somatic embryogenesis, and only one
plant is regenerated from each leaf explant to keep the regenerated
lines truly independent. Genomic DNA is extracted from the leaves of
each newly regenerated plant, which is used for both pooling and TAILPCR to identify plant sequences flanking the Tnt1 insert. The DNA pool
is available for reverse screening using a combination of Tnt1 and genespecific primers. The TAIL-PCR products are housed in the Tnt1 FST
database and are available for BLAST search. Seeds harvested from the
regenerated lines are germinated and grown in a greenhouse to bulk
seeds for distribution and screen for various mutant phenotypes.
980
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Tagged and TILLING Populations in Legumes
Figure 2. Some examples of root and nodule mutant
phenotypes identified by forward genetic screening
of a Tnt1-tagged M. truncatula population. Plants
were 6 weeks old at the time of screening and grown in
Perlite/sand mix supplemented with low-phosphorus
and low-nitrogen nutrient solution. A, A portion of a
wild-type (WT) R108 root with two nitrogen-fixing
nodules. The rest are mutant phenotypes. B, Very
dwarf whole root showing deformed main and lateral
root formation. C, Another type of severely dwarf root
showing almost the whole root system in which main
and lateral root development is arrested. D, Supernodulator showing an unusually high number of
nodules on a portion of normal root system. E,
Nonnodulator with a short root in which main and
lateral roots start expanding but further growth is
arrested. F, A nodule mutant on a portion of a normal
root in which a new root emerges from the nodule
apex.
vides more allelic series of mutants compared with
other insertional mutagenesis due to the higher density of Tnt1 insertions. The highest frequencies of
confirmed alleles found so far are for the nodulation
gene NIN (nine), the lamina gene STENOFOLIA
(eight), and the unifoliate gene SINGLE LEAFLET1
(six). Interestingly, no two alleles of the nine inserts in
the NIN gene (C. Pislariu and M. Udvardi, personal
communication) or the eight inserts in the STENOFOLIA
gene (M. Tadege and K. Mysore, unpublished data)
have been found to have insertions in the same physical location. The insertion site sequences within the
gene or between these two genes also do not show any
particular homology or specific pattern consistent with
the absence of a strong sequence-specific target site for
Tnt1 insertion.
To customize reverse screening, genomic DNA has
been extracted from 13,500 lines and is being pooled in
one dimension with superpools of 500 lines, smaller
pools, and mini pools that are convenient for PCRbased screening. Two pairs of one Tnt1-specific and one
gene-specific primer combination are used in a standard PCR screening to look for Tnt1 insertion in a gene
of interest. The screening starts by running the PCR
with the superpools. Once a positive signal is identified
in the superpool(s), the screening continues down to
the smallest pool until the individual line(s) containing
the insert is identified. So far, 20 superpools from 10,000
lines have been optimized for the community service.
FST sequencing is in progress to recover most of the
plant sequences flanking the Tnt1 insert. Tnt1-flanking
plant sequences are obtained by TAIL-PCR using a
combination of two or three nested Tnt1-specific
primers and one arbitrary primer. PCR products are
then cloned and sequenced. Sequences are deposited in
a Tnt1 FST database (currently 15,476 FST sequences)
that is equipped with GBrowser and BLAST search
functions and is publicly available (http://bioinfo4.
noble.org/mutant/database.php). Protocols for an efficient 454 sequencing platform are being optimized to
significantly increase the number of FSTs available to
the community. Seeds corresponding to the FSTs of
interest can be ordered online for a small handling
charge per line. Nondestructive phenotype screening
of the Tnt1 lines and reverse genetic screening of
DNA pools can be arranged by contacting K. Mysore
([email protected]) until an online service is established.
TILLING RESOURCES
The first plant TILLING service was developed for
Arabidopsis using LICOR sequencers (Till et al.,
2006a) at the Fred Hutchinson Cancer Research Center in Seattle following the pioneering work of J.G.
Henikoff and colleagues and is still in operation today
(http://tilling.fhcrc.org/). The first outside the United
States was for a legume model, L. japonicus. Lotus
TILLING was a collaborative venture between the
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Tadege et al.
Sainsbury Laboratory and the John Innes Centre in
Norwich (http://www.lotusjaponicus.org/tillingpages/
homepage.htm). Initially, an ABI377 gel-based platform was employed (Perry et al., 2003), but this was
superseded by LICOR 4300 instruments, which are
now the most commonly used. Similar platforms were
also set up by D. Cook (University of California, Davis)
and under the European Union Grain Legumes Integrated Project program for the partner model, M.
truncatula (http://www.glttp.com/products_services/
technical_services/genomic_resources_from_glip/
functional_genomics/medicago_tilling_platform),
and for pea (http://www.gl-ttp.com/products_services/
technical_services/genomic_resources_from_glip/
functional_genomics/pea_tilling_platform). Since then,
there have been several platforms established for a
range of legume species, including phenotype classifications for some (Table I). The L. japonicus service
currently operates as RevGenUK (http://revgenuk.jic.
ac.uk), which also carries out TILLING in M. truncatula
and Brassica rapa, all using a capillary electrophoresis
ABI3730 sequencing platform. For this platform, specialized protocols had to be developed to obtain sufficient lengths of sequence reads (Le Signor et al., 2009).
Improvements to all platforms are ongoing. For example, costs can be reduced on the LICOR instrument
using the dye Cy5 and internal labeling of primers
(K. Meksem and P. Gresshoff, personal communication).
Lotus TILLING established tools for both forward
and reverse genetic analyses. Arguably, it is the most
extensively used platform and has generated much
data. A structured population of M2 progeny of 4,904
EMS-mutagenized M1 embryos was generated in the
ecotype Gifu, but thematic populations were also
assembled (Perry et al., 2009) for development, starch
accumulation, and the nitrogen-fixing root nodule
symbiosis. The phenotypes (Perry et al., 2003) can be
interrogated via a Web-based database (http://data.
jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus/). Similar databases are available for other legumes (Table I). A new
population of approximately 3,500 lines in L. japonicus
ecotype MG20 is currently being assembled. The mutation load of the original population has been calculated to be one per 502 kb, equivalent to approximately
940 mutations per genome based on subsets of genes.
Lotus TILLING carried out mutation detection for 23
research groups across 11 countries and analyzed 158
targets. Interestingly, in examining these mutations in
detail, a 1:10 ratio between homozygous and heterozygous mutations in the M2 progeny was observed.
This result is in stark contrast to the 1:2 ratio observed
for Arabidopsis (Greene et al., 2003) and indicates an
unusual number of genetically effective cells in L.
japonicus producing up to six gametes. Furthermore,
by analyzing the distribution of amino acids that were
replaced by EMS mutagenesis in the missense and
nonsense alleles in functionally impaired mutant lines,
they revealed a significant bias for replacements of Gly
residues in the defective alleles. This may be explained
by the exceptional structural features of Gly, since Gly
allows peptide chains to adopt certain conformations
that are not possible once it has been replaced. TILLING
in legumes has been used largely for functional
genomics research to date. This has been either to
confirm, by generating additional alleles, a lesion in
forward screened mutants, especially those associated
with the Rhizobium-legume symbiosis, or to generate
unique mutants. As examples of the former, Perry et al.
Table I. Examples of current EMS mutagenesis resources for forward and TILLING reverse genetics in legumes
Mode: F, forward genetics; R, reverse genetics; S, service. Sites: CNR-IGV, Consiglio Nationale delle Richerche-Instituto do Genetica Vegetale;
INRA, L’Institut National de la Recherche Agronomique; JIC, John Innes Centre; SIU, Southern Illinois University; UGA, University of Georgia;
USDA-ARS, United States Department of Agriculture-Agricultural Research Service; WSU, Washington State University.
Species
a
Site
Arachis hypogaea
Cicer arietinum
F/R
F/R
UGA, Tifton
WSU, Pullman
Glycine max
F/R
SIU, Carbondale
Lotus japonicus
F
JIC, Norwich
S/R
JIC, Norwich
S/R
INRA, Evry
S/R
JIC, Norwich
INRA, Dijon
CNR-IGV, Perugia
USDA-ARS,
Puerto Rico
USDA-ARS, Idaho
INRA, Evry
Medicago
truncatula
Phaseolus
vulgaris
Pisum sativum
a
Mode
F/R
R
F
S/F/R
URL
Contact
Associated Refs.
[email protected]
[email protected]
http://www.soybeantilling.
org/index.jsp
http://data.jic.bbsrc.ac.uk/
cgi-bin/lotusjaponicus/
http://www.lotusjaponicus.org/;
http://revgenuk.jic.ac.uk/
http://www.versailles.inra.fr/urgv/
tilling.htm
http://revgenuk.jic.ac.uk/
http://www.igv.cnr.it/welcome/
http://urgv.evry.inra.fr/UTILLdb
[email protected]
Ramos et al. (2009)
Muehlbauer and
Rajesh (2008)
Cooper et al. (2008)
[email protected]
Perry et al. (2003)
[email protected]
Perry et al. (2003, 2009)
[email protected]
Le Signor et al. (2009)
[email protected]
Le Signor et al. (2009)
[email protected]
[email protected]
Porceddu et al. (2008)
Porch et al. (2009)
[email protected]
[email protected]
Dalmais et al. (2008)
Currently EcoTILLING (TILLING on ecotypes; Till et al., 2006b), with a full TILLING population under development.
982
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Tagged and TILLING Populations in Legumes
(2009) recently reported allelic series for 12 L. japonicus
genes involved in the symbiosis for use in structurefunction studies, and Hofer et al. (2009) used pea
TILLING to support the identification of the Tendrilless gene, whereas for the latter, Welham et al. (2009)
used TILLING alone to show that a cytosolic invertase
was crucial for normal plant growth and cellular
development but did not affect nodule function.
Since TILLING is readily applicable to most plant
species, it is especially suited to species where there are
few genomic resources and where insertion mutagenesis to create knockout mutants is difficult either
through a lack of appropriate elements or an inefficient
transformation system. Moreover, since it is a nongenetically modified (GM) technology, it is also especially desirable in those crops/countries where
application of GM technology is restricted. These advantages have facilitated its swift move from models to
legume crops and have meant that some crops have been
used in depth already. Pea was used as a pan-European
platform under the European Union Grain Legumes
Integrative Program (http://www.eugrainlegumes.
org/), and this has a direct link to the breeders and
other stakeholders under its Technology Transfer
Platform (http://www.gl-ttp.com/products_services/
technical_services/genomic_resources_from_glip/
functional_genomics). Soybean TILLING, established
with support from the U.S. Department of Agriculture
(USDA), has now targeted almost 130 amplicons across
4,600 lines (divided between ‘Forrest’ and ‘Williams82’
plants), one mutation on average occurring every
130 or 190 kb, respectively. The soybean platform has
extensive agronomic targets (K. Meksem, personal
communication), including low phytate, higher seed
yield and improved seed oil quality, improved
nodulation (based on the NARK mutants, in collaboration with P. Gresshoff and colleagues at the University
of Queensland), and improved resistance to cyst nematode.
Phaseolus, chickpea (Cicer arietinum), and lupin (Lupinus albus) TILLING are in different degrees of development. A population of Phaseolus ‘BAT93’ has
been established jointly by two groups (Porch et al.,
2009), and populations of U.S. market-class varieties
are being developed by V. Raboy (USDA-Agricultural
Research Service [ARS], Aberdeen, MD) and K. Cichy
(USDA-ARS, East Lansing, MI). These will be ready
for forward genetics screening in 2010. F.J. Muehlbauer
and P.N. Rajesh obtained 21,000 M1 plants of chickpea
(‘ICC12004’, a brown desi type) and have 2,855 lines
for TILLING to date with the aim of establishing a
population of 6,000 (P.N. Rajesh, personal communication). A subsample has been used to examine suitability for TILLING, and they found a mutation
frequency of one mutation per 165 kb, which is well
within the usable range. Some mutants have been
phenotyped in detail for vegetative (growth habit,
albino, leaf and stem structures, prostrate growth
habit, increased or decreased branching) and reproductive (flower color, petals missing or stunted, sta-
mens missing or stunted, pistils missing or stunted,
open flower, repetitive floral whorls, stunted flowers)
characters. Similarly, a TILLING population of 10,000
peanut (Arachis hypogaea) lines has been established,
and a subset was already used to investigate allergenicity (Ramos et al., 2009). At the Commonwealth
Scientific and Industrial Research Organization’s Division of Plant Industry, the development of populations of narrow-leafed lupin (Lupinus angustifolius) for
reverse genetics is under way (K. Singh, personal
communication).
BEYOND MUTAGENESIS
The prediction of Henikoff and Comai (2003) that
TILLING had the potential to become a standard
reverse genetic strategy for plant functional genomics
is well on its way to being realized. Its ease of use,
widespread applicability, relatively low cost (when
plant population costs are taken into account), and
ability to generate allelic series including knockouts
(albeit at a lower frequency than insertions) give it
advantages over other technologies. Its non-GM technology provides a further asset that has facilitated its
rapid move from model to crop legumes. Similarly, the
beauty of Tnt1 insertional mutagenesis in M. truncatula
in generating multiple knockouts with a high density
of allelic series will make it a very useful and handy
tool critical for gene function analysis. Since insertion
sites can be readily mapped using tags, libraries of
mutations can be created for browsing by the community. For TILLING this is not so easy, but one can
envisage that the use of next-generation sequencing
will come into play as costs diminish, so that it will be
more effective to use this technology to resequence
whole EMS-mutagenized populations and database
their full mutation landscapes. There are research efforts in this area currently for plants (e.g. http://tilling.
ucdavis.edu/index.php/TILLING-by-Sequencing).
ACKNOWLEDGMENTS
We thank all those researchers who provided us with unpublished and
prepublication data. We also thank Hee-Kyung Lee, Erin Hartwell, Janie
Gallaway, and Colleen Elles for their excellent technical assistance in
regenerating and maintaining the M. truncatula Tnt1 lines and Jillian Perry
for reading the manuscript.
Received July 1, 2009; accepted September 3, 2009; published September 9,
2009.
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