20.2 Mutagens and Identification
of the Mutations
Forward genetic screening for mutant phenotypes
followed by gene cloning using genetic mapping
has frequently and successfully been used for
identifying genetic components controlling a
wide variety of plant traits. Usually, the mutag-
enized population is single use, meaning that
mutations that do not affect the traits of interest
will be lost. In the post-genomic era, a large
demand for loss-of-function alleles of genes of
interest for hypothesis testing quickly became
apparent. Mutant collections comprising large
mutagenized plant populations complete with
information about all induced mutations in the
genome of each individual have proved the most
efficient way to meet this need, as evidenced by
the Arabidopsis T-DNA mutant collection
(Alonso et al. 2003 ).
There are three main combinations of muta-
gens and procedures for mutation characteriza-
tion in mutant collections (Table20.1). The most
widely used mutagens are chemicals, such as
EMS (ethyl methanesulfonate), that induce
nucleotide substitutions and small indels. These
mutations can be detected using a technique
called targeting induced local lesions in genomes
(TILLING), which relies on cleavage by single
strand-specific nucleases at sites where mis-
matches between wild-type and mutant sequen-
ces occur (McCallum et al. 2000 ). At the
moment, however, comprehensive characteriza-
tion of all mutations in an entire mutagenized
population has not been undertaken, probably
because it would require a very extensive
sequencing effort.
High-energy radiation is another common
way to mutagenize plants. Thefirst fast neutron
mutant population for reverse-genetic use was
established by Li et al. ( 2001 ). They employed a
simple PCR technique to detect deletions in a
genomic region of interest through identification
of amplicons smaller than those amplified from
wild-type alleles. It has been shown that genome-
wide array analysis allows detection of deletions
induced by fast neutron radiation in soybean
(Bolon et al. 2011 ). However, so far it has not
been feasible to identify the majority of the
deletions in the population because of the
excessive cost and the difficulty in identifying
smaller deletions.
Another type of mutagenesis relies on inser-
tion of DNA sequences. The advantage of using
DNA insertion for mutagenesis is the simplicity
of identifying the induced mutations, provided
that the sequence information for the inserted
DNA fragments is available. DNA fragment
insertion sites can then easily be determined by
sequencingflanking region amplicons. The sim-
plicity also makes it possible to comprehensively
characterize entire mutagenized populations
more efficiently than when using the alternative
methods described above. In most cases,
T-DNAs or transposable elements (TEs) are used
for generating the insertions. Both have been
used in Arabidopsis, and in all cases, exogenous
DNA fragments were used as mutagens. This
makes the resulting mutants transgenic, requiring
biocontainment precautions to be taken when
handling the plant material. Considering the
relatively small size of Arabidopsis plants and its
common use in controlled laboratory environ-
ments, this is not a major issue. However, in
many other plant species, including most crops,
the use of transgenic-based insertional mutagen-
esis is not attractive because of larger plant sizes
and recalcitrance to transformation. For these
reasons, comprehensive mutant collections in
other plant species are scarce, and there remains
a great need for such resources in most plant
research communities.Table 20.1 Mutagens and mutation detection methods
Mutagen Expected types of mutations Detection method
Chemical (e.g., EMS) Nucleotide substitutions, indels TILLING, sequencing
High-energy radiation (e.g., Fast neutron) Deletions, rearrangements PCR, DNA array, de-TILLING
Insertion tagging (e.g., TEs, T-DNA) Insertions Linker-assisted PCR222 E. Fukai et al.