Forwar and Reverse Genetics-1

After completion of the sequencing of the Arabidopsis
thaliana genome in 2000 (REF. 1), the plant biology

community faced the new challenge of assigning
biological functions to all of the genes in this 120 Mb
genome. Computational annotation of the genome
was initiated to predict the locations of the genes and
their basic structural elements (introns, exons
and putative regulatory sequences) and led to rapid
annotation of more than 25,000 Arabidopsis thaliana
genes
1
. Although extremely useful, such ab initio
annotation generates numerous inaccuracies at least
40% of gene predictions were subsequently found to
be erroneous
2,3
. Further refinement and validation of
the computational gene models and identification of
additional genes not predicted by gene-finding algo-
rithms has been achieved using various experimental
approaches
4,5
.
The main source of empirical information about
gene structure has been the capture and characteri-
zation of RNA transcripts (FIG. 1). A variety of high-
throughput methodologies have been successfully used
in A. thaliana, including ESTs and full-length cDNA
sequences
2,6,7
, whole-genome tiling microarrays
2
and
gene-expression arrays
2,8
, a massively parallel signature
sequence technique
9
, and serial analysis of gene expression
10,11
.
Combined, they have provided conclusive evidence
for the existence of thousands of new genes, as well as
complete gene-structure information for ~18,500 genes
(see The Arabidopsis Information Resource homepage).
At the time of completion of the genome sequence,
only ~10% of the 25,500 genes that were initially
predicted had an experimentally assigned function
1
.
Determination of the functions of the remaining
90% of genes presents a tremendous challenge, not
only because of the large number of genes to be
examined, but also because defining what constitutes
a gene is itself a complex problem
12
.
Recent improvements in genomic technology allow
genome-wide capture of some basic information, such
as the determination of gene or protein expression lev-
els using microarrays or shotgun mass spectrometry
1315
.
Although informative, these types of data alone are
typically not sufficient to define the function of a
gene, as by its very nature this information is largely
correlative. In fact, studies in yeast suggest that func-
tional inferences that are based on gene-expression
data can be misleading in many cases; for example,
Giaever et al. found that most mutants that showed
altered fitness in particular stress conditions cor-
responded to genes that were not transcriptionally
regulated by that type of stress
16
.
Mutant analysis provides an alternative and typi-
cally more reliable means to assign gene function
17
.
However, this phenotype-centric process, classically
known as forward genetics, typically is not suitable
for systematic genome-wide gene analysis, primarily
*North Carolina State
University, Department of
Genetics, Raleigh, North
Carolina 27695-7614, USA.
The Salk Institute for

Biological Studies, Plant
Biology and Genomic Analysis
Laboratories, La Jolla,
California 92037, USA.
Correspondence to J.M.A.
and J.R.E.
e-mails: jmalonso@unity.
ncsu.edu; ecker@salk.edu
doi:10.1038/nrg1893
Published online 6 June 2006
Whole-genome tiling
microarray
A high-density oligonucleotide
array that represents the
majority of DNA sequences of
an organisms genome.
Massively parallel signature
sequencing
A sequencing procedure that
allows the reading, in parallel,
of short sequence segments,
about 17 or 12 nt long, from
hundreds of thousands of
microbead-attached cDNAs.
Moving forward in reverse: genetic
technologies to enable genome-wide
phenomic screens in Arabidopsis
Jose M. Alonso* and Joseph R. Ecker
Abstract | Genome sequencing, in combination with various computational and

empirical approaches to sequence annotation, has made possible the identification
of more than 30,000 genes in Arabidopsis thaliana. Increasingly sophisticated genetic
tools are being developed with the long-term goal of understanding how the
coordinated activity of these genes gives rise to a complex organism. The
combination of classical forward genetics with recently developed genome-wide,
gene-indexed mutant collections is beginning to revolutionize the way in which gene
functions are studied in plants. High-throughput screens using these mutant
populations should provide a means to analyse plant gene functions the
phenome on a genomic scale.
REVI EWS
NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 1
Nature Reviews Genetics | AOP, published online 6 June 2006; doi:10.1038/nrg1893
2006 Nature Publishing Group

Forward genetics Reverse genetics
Genome sequence
Gene discovery
Gene functional annotation
Gene expression Mutant analysis
a Select a biological process
b Generate highly redundant mutant populations
c Screen for mutants with a desired phenotype
d Map and clone the gene responsible for the phenotype
a Select a gene or genes of interest
b Generate highly redundant mutant populations
c Develop and conduct sequence-based mutant screens
d Analyse the phenotype of the mutants
In silico prediction Proteinprotein interactions
ESTs Full-length cDNAs Tiling and expression arrays MPSS In silico gene prediction SAGE
Serial analysis of gene
expression
A technique that is used to
obtain short sequence tags
(typically 16 nt long) from large
numbers of cDNA clones by
cutting, concatemerizing and
finally sequencing cDNA
fragments.
Shotgun mass spectrometry
A bottom-up proteomics
approach that is used in the
identification of individual
components of a complex
protein mixture by the
identification of peptide
fragments on the basis of
their mass.
owing to the enormous effort required to identify
each gene responsible for a particular phenotype
18
.
In spite of improvements in the cloning of genes on
the basis of phenotype (such as the availability of
whole-genome sequences, large numbers of mapped
polymorphisms, and faster and cheaper genotyping
technologies), it can often take over a year for a
skilled scientist to move from a mutant to the affected
gene
19
(BOX 1).
Faster and easier ways to test hypotheses about
specific gene functions are expected in the post-
genome era. Various reverse genetic approaches have
been developed that leverage the flood of available
information from whole-genome sequences in order
to understand gene function. To identify a mutant
of interest, these technologies primarily rely on
predictions of function that are based on DNA
sequence homology
2024
. In the process of generating
tools for reverse genetics, large collections of gene-
indexed mutations have been created. The expected
improvement and expansion of these mutant collec-
tions in the near future promises to aid biologists in
meeting the main challenge of the post-genome era:
the systematic analysis of the functions of all genes in a
genome.
In this review, we discuss the challenges associated
with characterizing gene function in the post-genome
era in the reference plant A. thaliana. Classical and
more recent genome-wide experimental approaches
are described and compared in the context of the
complexities of systematic studies of gene function in
this model organism. We conclude with a discussion
of how several new and up-and-coming technologies
could profoundly affect this field in the near future.
Perturbing gene activity
Random mutagenesis. In both forward and reverse
genetic approaches, the function of a gene is typically
assigned to a specific biological process by analysing the
phenotypic consequences of altering that genes activity.
A wide variety of physical, chemical and biological agents
can be used to create genetic alterations in A. thaliana
17
,
each of which has a different efficiency (the number of
events per genome that are inherited in a population that
has been mutagenized under standard conditions) and
molecular outcome (ranging from a single nucleotide
substitution to an insertion or a rearrangement of large
chromosomal regions
25
).
Fast neutrons, -radiation and carbon ions are most
commonly used for mutagenesis in A. thaliana
25,26
. In
general, the efficiency of high-energy ionizing radiation
is intermediate, and the induced alterations typically
include chromosomal deletions and rearrangements,
although point mutations are not uncommon
25,26
.
Although high mutagenicity means that fewer plants
need to be screened to identify mutants with the desired
phenotype, it also means that numerous unrelated
mutations must be removed before the mutant pheno-
types can be analysed (typically, by several rounds of
backcrossing to the parental non-mutagenized line).
The nature of the damage that is caused by mutagen-
esis determines the functional class of genetic alterations
that are produced. Deletions, insertions and rearrange-
ments are more likely to result in loss-of-function alleles,
whereas point mutations can lead to a broader range of
effects, including hypomorphic, hypermorphic and neo-
morphic effects (that is, alleles of reduced, enhanced or
novel gene function, in corresponding order). The nature
of the damage also has implications for the experimental
Figure 1 | From genome sequence to gene function. Steps and experimental approaches that are used in the
functional annotation of the genome. MPSS, massively parallel signature sequencing; SAGE, serial analysis of
gene expression.
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f Catalogue the mutants in the collection
a Select a biological process b Select a gene or genes
Forward genetics Reverse genetics
d Generate a collection of mutants or tools
Map insertions in the
sequenced genome
Sequence
insertion sites
e Screen a large number of
mutagenized M2 plants
c Generate a random and highly
redundant mutant population
h Select mutants in the gene or genes
of interest
g Map and clone the mutation
EMS mutagenesis Plant transformation
A
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Box 1 | Forward versus reverse genetic approaches

The selection of a biological process is the first step in
forward genetics, illustrated in panel a by the response to
ethylene. Three-day-old Arabidopsis thaliana seedlings that
were grown in the dark in the absence (left panel) or
presence (right panel) of ethylene are shown. The clear
difference between treated and untreated plants is an
excellent morphological trait for use in a screen. By
contrast, a typical reverse genetic screen starts with a set
of genes of known sequence that are of particular interest
(in panel b a hypothetical family of genes (generic
Arabidopsis nomenclature is used) is selected on the basis
of their sequence similarity).
The next step in both forward and reverse genetics is to
generate a population of mutants. After mutagen
treatment (panel c), seeds are germinated in soil and
grown in families (with ~1,000 individual mutagenized
plants per family) to maturity to produce the M1
generation. Their progeny (M2) are typically used for
phenotypic screening. For transferred DNA (T-DNA)
mutagenesis, adult plants are transformed by submerging
the floral buds in a suspension of Agrobacterium
tumefaciens cells that harbour an appropriate T-DNA-
containing vector (panel d). The seeds of these plants are
grown in the presence of a selective medium, so that only
the plants expressing the resistance marker gene that is
supplied by the T-DNA survive. Each transformant (T1)
results from an independent transformation event and is
hemizygous for one or more insertion. The progeny of a
selfed T1 plant (T2 family) is composed of a segregating
population of wild-type plants, and hemizygous and
homozygous mutants. The T2 generation is usually used in
screens. Panel e shows a typical phenotypic forward
genetic screen of M2 seeds. A mutant resistant to the
hormone can be easily identified among thousands of
normally responding plants.
In a reverse genetic screen, once the T-DNA mutants are
obtained, two different approaches can be used: T-DNA
pools are screened by PCR (FIG. 2) or the insertion sites are
mapped in individual T2 families (shown in panel f). To map
the insertion sites genomic DNAs are obtained from
individual T2 families, and DNA fragments flanking the
insertion sites are amplified by PCR and sequenced.
Sequencing of hundreds of thousands of insertion sites
results in a gene-indexed catalogue of mutants.
The last step in a forward genetic approach is the
identification of the gene that is responsible for the mutant
phenotype. Panel g illustrates the series of mapping steps
required to determine the position of the mutation in the
genome, starting from a large chromosomal region and
zooming in to the gene level. Sequencing and
complementation studies are required for conclusive
mutant identification. The reverse genetics counterpart is
extremely fast and easy. Once a catalogue of gene-indexed
mutants has been obtained, it can be searched for the
presence of mutants in any gene of interest. Once found,
seeds of the corresponding mutant(s) can be ordered from
one of the public stock centres (panel h). It is important to
point out, however, that in the reverse screening approach,
this step is the starting point of a sometimes tedious search
for the mutant phenotype, whereas in the forward genetic
approach, this step is already accomplished (panel a).
*Requires one skilled person. **Requires two to five skilled
people. ***Requires five to ten skilled people. EMS, ethyl
methanesulphonate.
REVI EWS

a
b
c
Chemical agents
(for example, EMS)
Biological agents
(Transposons and
T-DNA)
Use in reverse genetic approaches Mutagen
+
+
+
Large range of mutations
including loss and gain of function
Very high efciency with hundreds
of mutations per genome
Difcult to nd the
mutation in the genome
Medium efciency
Laborious identication
of the mutations
Easy identication of the
mutations
Main characteristics
A
C
A
T
G
C
A
T
G
C
T-DNA
Point mutations
Big insertions, deletions
and rearrangements
Mostly loss-of-function mutants
Low efciency
Insertion of specic DNA sequences
Physical agents
(Fast neutrons,
X-rays and
accelerated ions)
Mostly loss-of-function mutants
PCR-based screening of insertional mutants
TILLING
Deleteagene
strategy that is best suited to identifying the genomic loca-
tion of the mutation. Methods such as Deleteagene
23
(see
below for details), subtractive hybridization (illustrated
by the cloning of a gibberellin biosynthetic mutant ga1
using a fast neutron allele ga1-3 (REF. 27)), or high-density
microarray-based mapping
28,29
take advantage of chro-
mosomal deletions for rapid gene identification. Other
types of DNA alteration, such as point mutations, require
different and, traditionally, more tedious experimental
approaches (FIG. 2).
Chemical agents, such as ethyl methanesulphonate
(EMS) and nitrosomethylurea (NMU), are extremely
efficient mutagens in A. thaliana. Under optimal con-
ditions, EMS treatment of seeds can generate about
400 mutations per genome, compared with an average
of 1.5 insertions per transferred DNA (T-DNA) mutant
(see below for details)
30,31
. Owing to their high efficiency,
chemical agents are the first choice in exploratory
mutagenesis, where the frequency of specific mutant
phenotypes needs to be evaluated
32
. Chemical agents
generate a broad range of DNA alterations; these are
predominantly single base-pair substitutions, but also
include small insertions and deletions, as demonstrated
in a recent large-scale study of more than 1,900 EMS-
induced mutations in A. thaliana
31,33
. Importantly, the
distribution of EMS-induced mutations is unbiased. But
chemical mutagenesis has one main drawback finding
the DNA alteration that is responsible for a mutant
phenotype, which is typically a single base change, is
difficult in a large genome. This handicap of chemical
Figure 2 | Generation of genetic diversity. a | Point mutations can be identified using TILLING (targeting induced local
lesions in genomes). DNAs from pools of EMS (ethyl methanesulphonate)-mutagenized plants are PCR-amplified using
primers that are specific for the gene of interest. Because each primer is labelled with a different fluorescent dye, both
DNA strands can be visualized simultaneously. Amplified DNA products are denatured and then allowed to renature,
leading to the formation of homodimers and heterodimers between the wild-type and mutant DNA (only heterodimers
are shown for simplicity). Mismatches are recognized and cleaved by the mismatch-specific celery nuclease CELI. The
presence of a mutation in a pool is then determined by separating the products of the CELI reaction in a denaturing gel.
The presence of two different size bands that are labelled with different fluorescent dyes in a given pool indicates the
presence of a mutation in the gene of interest. The specific sizes of the bands provide information about the position of
the mutation in the gene. Mutations are confirmed by sequencing. b | Identification of deletions from pools of plants
mutagenized with fast neutrons. DNA from the pools of mutants is PCR-amplified using primers that flank the gene of
interest. The amplification products are then analysed by gel electrophoresis. In some pools, a band that is smaller than
that predicted for the wild type (marked with an arrow) can be detected, which indicates the presence of a deletion in
one of the plants in that pool. Secondary screening of individual plants in the pool confirms the presence of the
mutation and identifies individual mutants. c | The presence of a transferred DNA (T-DNA) insertion in a gene can be
detected using primers that are specific for the gene of interest and for the T-DNA. DNA from mutant plants is used as a
template for PCR amplification. The presence of an amplification product (marked with an arrow) indicates the possible
existence of an insertion in or near the gene of interest. Individual plants from the pool are tested to identify the mutant
line. Alternatively, the insertion sites can be systematically identified, as indicated in BOX 1.
REVI EWS

Mismatch-repair detection
on tag arrays
A hybridization-based
technique that combines a
bacterial selection assay and
microarray analysis to rapidly
scan large genomic regions for
the presence of DNA
polymorphisms.
mutagenesis, which is the method of choice in forward
genetic screens, is even more serious when applied in the
reverse genetic mode, where the detection of the sequence
alteration is the only way of finding the mutant. However,
the development of highly sensitive methodologies for
point-mutation detection such as targeting induced
local lesions in genomes (TILLING) (see below for
details)
31,34,35
is now allowing the incorporation of the
power of chemical mutagenesis into the reverse genetic
toolbox. In addition, new methods for efficient genome-
wide detection of point mutations, such as mismatch-repair
detection on tag arrays, are appearing on the horizon
36
.
Biological agents that mediate the transfer of DNA
or RNA molecules into plant cells can also be used as
mutagens. Depending on whether the DNA or RNA is
used to disrupt the sequence of a gene or to alter its activ-
ity indirectly, one can differentiate between insertional
and gene-replacement mutagenesis
37
versus overexpres-
sion and gene-silencing methods
38,39
. In either case, the
first step is the introduction of an engineered DNA, or
sometimes RNA, molecule into living cells. The most
common method of introducing a DNA molecule into
plant cells relies on Agrobacterium tumefaciens as a shut-
tle
40
. These soil-born bacteria can infect plant cells and
transfer specific sequences that are contained in their
Ti plasmid into the plant genome
40,41
. Most of this plas-
mids sequence can be replaced without affecting transfer
efficiency
42
. Several other types of plant-associated
bacteria, such as Rhizobium spp., Sinorhizobium meliloti
and Mesorhizobium loti, are also capable of horizontal
transmission and integration of plasmid DNA into plant
chromosomes, opening up the possibility of gene trans-
fer to plant species that are resistant to Agrobacterium-
mediated transformation
43
. It is also possible to use DNA
or RNA viruses to deliver genetic information into plant
cells
44
. In this case, the new genetic material remains
associated with the viral genome, which limits its use
in the transient alteration of gene function (TABLE 1; see
below for further discussion). Alternatively, cells can be
bombarded with DNA-coated microparticles
45
.
Arabidopsis thaliana can be stably transformed by sub-
merging floral buds into a suspension of the appropriate
Agrobacterium strain. Owing to the simplicity and ease of
this floral dip method
46
, Agrobacterium-mediated trans-
formation has been widely used in gene-function studies.
In the simplest case, a selectable marker, such as a herbicide
resistance gene or an antibiotic resistance gene, is inserted
into the T-DNA, in addition to the T-DNA left and right
border sequences that mediate the T-strand movement
from the bacterial plasmid to the plant cell nucleus
41
. If
the T-DNA inserts within the boundaries of a gene, it
can drastically alter, and in many cases completely abol-
ish, gene function
47
. Such simple Ti plasmids that carry
selectable markers have often been the preferred type of
vector in the generation of large T-DNA mutant collections
that are designed exclusively to yield knockouts
30,48
.
Table 1 | Methods used for reverse genetics in plants
Method Advantages Disadvantages
Homologous
recombination*
Allows for exact replacement or modification of the targeted gene
Highly specific to the target gene (no off-target effects)
Results in stable mutations
Very low efficiency
Low throughput
Gene silencing
Possibility of generating allelic series, allowing the study of

essential genes
Possibility of restricting the alterations to specific tissues or
developmental stages
Study of gene families with high degree of functional redundancy
Can be adapted for high-throughput screens
The degree of gene silencing is unpredictable
Risk of off-target effects
Instability of phenotypes
Ectopic
expression
Similar to gene silencing

Allows for the analysis of gain-of-function alleles
Can be adapted for high-throughput screens
Similar to gene silencing
Possibility of generating misleading neomorphs
Its use is limited to transformable species
Zinc-finger
nucleases*
Highly specific
Low throughput
Its use is limited to transformable species
TILLING
Allows the identification of loss-of-function alleles, hypomorphs

and gain-of-function alleles
Can be used in non-transformable species
Based on random mutagenesis, so the desired mutation
might never be found
Low to medium throughput
Deleteagene* Allows the identification of two or more genes in close proximity
Can be used in non-transformable species
Based on random mutagenesis, so the desired mutation
might never be found
Limited to loss-of-function mutations
Low to medium throughput
Insertional
mutagenesis
High throughput
Can be adapted for both loss-of-function and gain-of-function
studies
Few unwanted mutations
Based on random (T-DNA) or non-targeted (transposon)
mutagenesis, so the desired mutation might never be found
Cannot be used to study tandemly repeated genes (T-DNA
mutagenesis)
Only limited information can be obtained for essential
genes
*Each of these approaches has so far been of limited practical use, with fewer than 10 successful examples reported.
Each of these approaches was used as a tool

to study gene function in more than 50 publications.
There are more than 500 publications that used this technique. T-DNA, transferred DNA.
REVI EWS

loxP
A sequence that is specifically
recognized by the Cre
recombinase from the
bacteriophage P1. Cre
catalyses the recombination
between two loxP sequences.
Random insertional mutagenesis can be used in
many other ways. T-DNA or transposons can carry pro-
moterless reporter genes (such as -glucuronidase, GFP
or luciferase), which can trap the regulatory sequences
of the tagged gene
49,50
as a result, the reporter gene
will display the original expression patterns of the tagged
gene
49
. Promoter trap lines provide an excellent tool for
studying gene-expression patterns
49
. Systematic analy-
sis of trap lines provides molecular markers that can be
used to monitor in vivo developmental processes, such
as flower
51
or root
52
morphogenesis, responses to stress
53

and circadian rhythms
54
.
Owing to its disruptive nature, T-DNA mutagen-
esis is commonly associated with loss-of-function or
hypomorphic mutations. However, it can be adapted
to generate gain-of-function alleles by activation
tagging
38
. To achieve this, several copies of a strong tran-
scriptional enhancer are introduced into the T-DNA.
On integration, the enhancers stimulate the transcrip-
tion of a nearby gene and cause its ectopic expression.
Assigning biological function to a gene on the basis of
the phenotypic effects of its overexpression should be
done with caution, as illustrated by the initial assign-
ment of the histidine kinase CKI1 (CYTOKININ-
INDEPENDENT 1) as a putative receptor for the
hormone cytokinin
55
. Subsequent studies have not
only failed to confirm the role of CKI1 in cytokinin
reception, but instead showed that three histidine
kinases that are related to CKI1 (CRE1 (CYTOKININ
RESPONSE 1; also known as WOL), AHK2 and AHK3
(ARABIDOPSIS HISTIDINE KINASE 2 and 3)) are
the bona fide cytokinin receptors
56
. Nevertheless, there
are many examples that highlight the utility of activa-
tion tagging in gene-function studies
5760
, especially in
plant genomes in which high levels of gene duplication
and compensatory mechanisms make the analysis of
loss-of-function mutations challenging
6163
.
In even more sophisticated approaches, transposons
can be introduced into the plant genome using T-DNA-
mediated transformation. Once inserted, the transposon
can hop from one chromosomal location to another, as
long as an active transposase is present, with the potential
of creating mutations at both the landing and excision
sites
64
. Although most transposons tend to hop to linked
sites, a strategy has been devised to select for transposi-
tions that land at unlinked loci
65
. The mobile element
and the T-DNA integration site launch pad have been
engineered to harbour positive and negative selectable
markers, respectively. Plants that are able to grow in the
presence of both the positive and negative selection should
be greatly enriched for unlinked transposition events.
In most cases, it is desirable to fix the insertions after
a single hop, so that the phenotype of a stable mutant
can be studied. The most common way to regulate
transposon activity is to introduce both transposon
and transposase into the same plant (typically by co-
transforming plants with T-DNA constructs bearing
both
66
or by crossing a transposon-harbouring plant
with a plant harbouring an active transposase
67
) and
then to separate them (usually by crossing) to stabilize
the new mutations. Typically, once the transposon
has been allowed to jump, plants with new insertions
that do not contain an active transposase are selected
from the progeny of the cross to create stable mutant
(transposant) lines. Therefore, although the generation
of large numbers of transposon insertions is straightfor-
ward, considerable effort is required to select plants that
have stable insertions
65
.
The mobile properties of transposons have been
exploited in several other applications. For example,
reversion of the mutant phenotype after remobilization
of a transposon has been used to prove causal relation-
ships between a gene and a mutant phenotype
49,68,69
.
Transposon remobilization has also been used to gener-
ate sectors of heterozygous cells in a homozygous mutant
plant to determine whether or not a phenotype is cell
autonomous
49,70,71
. Transposons can also be engineered
to contain simple selectable markers, more elaborate
gene traps or transcriptional enhancer systems
49,72
.
A recently developed application takes advantage
of the predominance of local transposition to study
loss of function within gene families
73
. Approximately
17% of the predicted genes in A. thaliana are tandemly
repeated
1
. Functional analysis of tandemly repeated
genes is problematic because generating double mutants
of tightly linked loci by crossing is difficult. Local trans-
position, together with the high frequency of DNA
lesions at the site of transposon excision, allow for the
simultaneous disruption of several genetically adjacent
loci
64,74
. Furthermore, a specialized T-DNA system
composed of three elements a transposon, two loxP
recombination sites (one inside the transposon and one
in the immobile part of the T-DNA), and an inducible
loxP recombinase has been developed to generate
deletions of selected genomic regions
73
. Using a simi-
lar approach, Krysan and colleagues have generated a
population of ~10,000 randomly inserted loxP T-DNAs
for which the insertions sites have been determined (see
the New Wisconsin T-DNA Lines pDsLox Vector
web site). Once transposition is activated in a selected
line (by crossing in an active transposase) the recombi-
nase is activated, which leads to permanent removal of
the genomic region that is contained between the loxP
elements in the transposon and the immobile part of
the T-DNA. This strategy could be useful for study-
ing functionally redundant genes that are located in
adjacent chromosomal locations, which is a common
phenomenon in A. thaliana.
The aforementioned approaches, all of which pro-
duce lesions in unpredictable chromosomal locations,
can be used in forward and reverse genetic approaches.
There are, however, three main requirements that must
be met for a collection of random mutations to be
efficiently used in a reverse genetic screen. First, the
number of mutations in the collection should exceed
(by five to tenfold) the number of genes in the genome.
This redundancy is necessary to ensure that mutations
in a particular gene will be found with a sufficiently
high probability. Second, each individual plant in
the mutagenized population should be catalogued,
propagated and pooled so that it can then be effectively
screened. Third, and perhaps most important, a DNA
REVI EWS

RNA interference
A form of gene silencing in
which dsRNA induces the
degradation of homologous
endogenous mRNA transcripts,
thereby mimicking the effect of
reduction, or loss, of gene
activity.
Co-suppression
Silencing the expression of an
endogenous gene, which is
caused by the expression of a
transgene bearing high levels
of sequence identity with the
endogenous gene.
Post-transcriptional gene
silencing
Refers to the general
mechanisms that are involved
in gene silencing at the
post-transcriptional level
independently of the initial
silencing agent, that is,
double-stranded, antisense
or sense RNA.
Small interfering RNA
2025 nucleotide long RNA
molecules that are generated
during the post-transcriptional
gene-silencing process and
act as key determinants of
the sequence specificity of the
silencing mechanism.
MicroRNA genes
Genome-encoded or artificial
genes; the RNAs that are
transcribed from them are
processed to generate small
RNA fragments (2025 nt
long) that target specific
mRNAs for degradation or
inhibit their translation into
proteins.
sequence-based screening approach needs to be devel-
oped that is sensitive enough for a single plant with a
specific sequence alteration to be detected within a pool
of wild-type individuals. Different types of DNA lesion
(deletions, insertions and point mutations) require
different screening methodologies (see below).
Targeted mutagenesis. True directed mutagen-
esis approaches, in which the researcher chooses the
gene to be perturbed, are widely used in A. thaliana.
The most elegant and precise targeted mutagenesis
approach relies on homologous recombination to tar-
get foreign DNA to homologous sequences in the host
genome. Although a few successful attempts have been
reported in plants
75,76
, the extremely low frequencies
of recombination events make this approach ineffec-
tive for gene-function studies in A. thaliana (and in
most other eukaryotes)
77
. Improved selection strate-
gies and a better understanding of the basic molecular
mechanisms involved in homologous recombination
indicate that targeted gene replacement could become
a reality in the near future
78,79
. For example, Shaked
and co-workers
80
recently found that overexpression
of a yeast SWI/SNF chromatin remodelling enzyme,
RAD54, in A. thaliana resulted in a 27-fold increase
in gene targeting. If these results can be reproduced
for other genes, this improvement in efficiency, in
combination with stringent selection procedures, will
make gene targeting a routine approach in Arabidopsis
research. Meanwhile, alternative approaches have been
developed to alter the expression of selected genes.
There are two main variants of directed mutagenesis
(or methods of alteration of gene expression): gene-
silencing approaches and zinc-finger nucleases, which
are still in an experimental phase
81
. In these strategies,
specific sequences that are unique for each gene to be
disrupted must be engineered in vitro and then intro-
duced into the plant. Agrobacterium tumefaciens
40
,
viruses
44
or particle bombardment
45
have all been used
to mediate the transfer of such sequences.
First described in plants
82,83
, gene silencing seems to
have evolved as a defence mechanism in most eukaryo-
tes against viruses and active endogenous transposons
44
.
This mechanism, also called RNA interference (RNAi),
co-suppression or post-transcriptional gene silencing, can
be exploited for attenuating gene expression by intro-
ducing specific sequences into the plant cell. Once a
dsRNA of choice is expressed, a silencing signal is gen-
erated that can spread throughout the plant, causing
a reduction in the transcript levels of the endogenous
gene or genes in a sequence-specific manner
39
. In con-
trast with insertional mutagenesis, RNAi lines typically
show a wide range of effects on gene expression, from
complete inhibition to no reduction
39
. Although this
approach allows the analysis of allelic series that could
be important in the study of essential genes, the vari-
ability of the silencing effects in different plants and
tissues, and even between genes or gene constructs,
complicates the interpretation of results and neces-
sitates the analysis of several independent RNAi lines.
Another drawback stems from the fact that during the
processing of the dsRNA large numbers of different
small interfering RNAs (siRNAs) can be generated, mak-
ing it difficult to predict and therefore avoid off-target
effects
84
. Therefore, the sequences that are used to gen-
erate the dsRNA must be carefully selected, because
even stretches as short as 1011 nucleotides that are
homologous between the RNA and an endogenous
sequence can result in silencing
84
.
A possible solution to some of the problems asso-
ciated with using siRNAs might come from the use
of microRNAs (miRNAs)
85
. miRNAs can regulate the
expression of genes to which they are only partially
complementary, making the identification of all poten-
tial miRNA targets challenging. However, although
such sequence promiscuity has been found for both
plant and animal miRNA targets, genome-wide studies
in A. thaliana
8
indicate that plant miRNAs have fewer
targets than animals, perhaps as a result of tighter
mechanisms governing target recognition. Recent
genome-wide studies of the effects of several artificial
miRNAs (amiRNAs) on gene expression support this
hypothesis and identify key sequence determinants that
underlie miRNA specificity in plants
86
. It should now
be possible to design amiRNAs that selectively target
genes of interest, making this technology an excellent
tool for dissecting the function of essential genes. Most
importantly, these types of technology (amiRNA and
RNAi) will facilitate the functional characterization of a
potentially large number of Arabidopsis genes that have
overlapping or partially redundant functions by making
RNA molecules that are specific to a family of related
genes rather than to a single gene
8789
.
One important obstacle to a more widespread use
of the sequence-directed mutagenesis approaches
described above is that altering the function of the
targeted gene relies on the activity of a transgene. The
interpretation of these studies is also complicated by
the fact that transgene expression can vary markedly
from one plant to another and from one generation to
the next
90
. Recently, a novel sequence-directed muta-
genesis methodology has been described that uses
zinc-finger proteins to alter the sequence of a targeted
gene and, therefore, results in stable mutations
81
. Zinc-
finger proteins that recognize specific DNA sequences
can be engineered. Specific sequences can be cleaved
by combining these zinc-finger domains with a dsDNA
endonuclease in a single protein
91
. It has recently been
shown that the expression of a sequence-specific zinc-
finger nuclease in A. thaliana generates mutations
(deletions and insertions) in the target gene in planta
81
.
Stable mutations in the gene of choice can be isolated
because the permanent DNA damage that is inflicted
by the nuclease is heritable
81
. The large battery of well-
characterized zinc fingers, each with different and
specific DNA-recognition sequences, should allow the
use of this methodology to target almost any gene in
the A. thaliana genome. Chromosome breaks that are
induced in this way can enhance the rate of homolo-
gous recombination in specified target locations
92
,
opening another door to the future use of homologous
recombination in plants.
REVI EWS

Ectopic expression. In addition to triggering silenc-
ing, gene-specific sequences can be introduced into
the plant genome to enhance gene expression. In the
simplest case, a complete gene, including its regulatory
and coding regions, is introduced by Agrobacterium-
mediated transformation into the plant genome. This
approach is typically used in complementation assays,
in which a loss-of-function mutant is transformed with
the wild-type gene to confirm the phenotypegenotype
relationship or to study the effect of gene dosage.
In another application, the regulatory region of one
gene is fused with the coding region of another. Strong
constitutive promoters, such as cauliflower mosaic virus
35S (CaMV35S), are usually preferred in initial studies in
which the objective is to obtain general functional infor-
mation
93,94
. In other cases, it is often desirable to analyse
the effects of ectopic activation of a gene of interest in a
specific cell type or developmental stage. This laborious
process is facilitated by use of the GAL4GFP enhancer
trap system, which was initially developed in Drosophila
melanogaster
87
and later adapted for A. thaliana
88,89
. In
this approach, wild-type plants are transformed with a
trap construct that harbours a selectable marker and two
other genes: a promoterless gene that encodes the yeast
transcription factor GAL4, and a reporter gene (typically
GFP), which is under the control of a minimal promoter
and a UAS (upstream activating sequence), which is
recognized by the GAL4 transcription factor. When the
T-DNA that harbours the reporter construct lands in a
gene, the regulatory sequences of that native gene are
hijacked by the T-DNA and used to drive the expres-
sion of GAL4 in a manner characteristic of the particular
trapped gene promoter. The GAL4 transcription factor
will bind to the UAS and activate the expression of GFP
or any other gene that is placed under the control of the
UAS. By monitoring GFP fluorescence, one can easily
determine where and when GAL4 is expressed in a par-
ticular trap line. A catalogue of lines that display specific
GAL4GFP expression patterns (GAL4GFP trap lines)
can be generated by crossing a characterized GAL4GFP
line to a plant in which the transgene of interest is under
the control of the UAS promoter. Although such col-
lections have been generated, the frequent loss of GFP
expression in the trap lines, which is due to silencing in
later generations, limits their utility. It might be possible
to alleviate such problems by using promoters other than
CaMV35S to drive GAL4 expression, which could result
in less frequent activation of the cellular gene-silencing
machinery.
With this tremendous battery of tools at hand, three
main strategies can be used to study gene function:
classical forward genetics, classical reverse genetics and
systematic reverse genetics.
Classical forward genetics
Forward genetics has been a powerful approach to study-
ing the genetic components of almost every signalling
or biochemical process in A. thaliana
95,96
. One classical
example is the identification of mutants that express
the nuclear chlorophyll a/b binding protein (CAB)
gene in the absence of functional chloroplasts to study
nucleuschloroplast communication
97
. Another involves
forward screens for mutations that affect specific and
marked morphological changes induced in dark-grown
seedlings by ethylene to uncover a large number of
ethylene-related loci that are involved in either bio-
synthesis, perception, signalling or response to this
gaseous plant hormone
98
. In another example, the circa-
dian cycling of CAB gene expression has been exploited
to develop sensitive molecular markers to study biologi-
cal rhythms in plants
99
. By fusing a CAB promoter with a
luciferase reporter gene, Kay et al. were able to closely
monitor the circadian oscillations in planta, allowing
them to isolate mutants with altered circadian rhythmic-
ity
99
. High reproducibility, sensitivity and specificity,
together with the potential for high throughput, are the
main desirable characteristics of a phenotype in forward
genetic screens. Commercial and community resources
such as Lehle Seeds, Arabidopsis Biological Resource
Center Stocks, and the Nottingham Arabidopsis Stock
Center now provide different types of ready-to-screen
mutagenized seed, including lines that have been muta-
genized using EMS, fast neutrons, T-DNA and trans-
posons. Most have been generated in a limited number
of widely used genetic backgrounds (such as Columbia,
Wassilewskija and Landsberg erecta), rendering these
established collections unsuitable for highly special-
ized screens in which the use of a particular molecular
reporter or mutant background is desired. Therefore, indi-
vidual research groups often have to generate their own
mutagenized plant populations.
One of the most attractive features of forward genetic
approaches is that they represent truly unbiased gene-
discovery processes no preconceived idea about the
nature of the gene involved in the process is required.
Reverse genetics
The large amount of sequence information that has
been generated by genome projects for A. thaliana
1

and other plant species
62,63
, together with the imple-
mentation of genome-wide approaches to study gene
expression, proteinprotein interactions and other
aspects of plant biology, have resulted in an increased
interest in reverse genetic methodologies. Numerous
examples highlight the recent success of these reverse
genetic approaches. Analysis of multigenic families, in
which functional redundancy has made forward genetic
approaches difficult, has particularly benefited from
reverse genetic approaches. Entire gene families have
been knocked out, uncovering overlapping and specific
functions among their members
100102
. If the size of a
gene family is too large to disable all of its members,
criteria other than sequence similarity are often applied
to investigate their functions. For example, a combina-
tion of sequence comparison and expression profiling
was used to identify, out of over a hundred related
genes, a small subfamily of four transcription factors
that are involved in the early response to ethylene
30
.
Alternatively, approaches based on gene silencing can,
in principle, be used to alter the expression of several
members of a gene family. However, the utility of this
approach is yet to be demonstrated.
REVI EWS

Saturation mutagenesis
Mutagenesis as a result of
which there should be a 100%
probability of identifying at
least one mutation in any given
gene.
Thermal asymmetrical
interlaced PCR
A PCR-based strategy that
utilizes nested primers, which
are complementary to a
known DNA sequence, and
degenerate primers to amplify
the unknown flanking DNA
regions.
Adaptor ligation PCR
A PCR-based approach that is
used to determine the
sequence flanking a DNA
region of known sequence. It
utilizes two sets of nested
primers and a DNA adaptor
that, after its ligation to
fragmented DNA, facilitates
the amplification of the desired
DNA region.
There are two main approaches to finding mutations
within a gene on the basis of its DNA sequence: using
one of the targeted techniques such as RNAi or ectopic
expression, or screening a collection of randomly gen-
erated mutants for a knockout. The ease of producing
large collections of random mutants, the genetic stabil-
ity of the resulting mutations, and the recent develop-
ment of high-throughput sequence-based screening
strategies such as TILLING and Deletagene (see below)
have made screening for knockouts an essential tool in
reverse genetics
103
. Perhaps, the most important single
prerequisite for choosing a collection of randomly gener-
ated mutations for a reverse genetic screen is the avail-
ability of highly efficient screening methods to identify
sequence alterations in genomic DNA. The number of
individual mutants that are screened tends to be very
high (in some cases, in the order of tens of thousands),
making it necessary to pool individual mutants before
testing for the presence of the mutation of interest. The
optimal strategy for screening these DNA pools depends
on the type of mutagen used or, more specifically, the
nature of the DNA lesion.
TILLING. TILLING has been developed to identify
mutations in EMS-mutagenized populations
34,35
.
Detection relies on the amplification of the gene of
interest from pooled DNA. After PCR amplification,
the DNA is denatured, renatured and then digested
with the mismatch-specific celery nuclease CELI,
which recognizes base-pair mismatches
104
. The exist-
ence of a mutation in a particular DNA pool can be
identified by the presence of two DNA fragments on
a denaturing acrylamide gel which is a result of
nuclease activity at the site of the mismatch between
the wild-type and mutant heteroduplexed DNA frag-
ments (FIG. 2a). DNA from the individual mutants that
comprise the positive DNA pool is screened to identify
the individual plant that carries the mutation. The
main advantage of this strategy is that it integrates a
reverse genetic approach with the benefits of chemical
mutagenesis, which enables a wide range of genetic
alterations to be produced: not only loss-of-function
alleles, but also hypomorphic, hypermorphic and neo-
morphic mutations
105
. Nonetheless, as this technology
remains relatively labour intensive, it is typically rel-
egated to second choice and is usually relied on when
sequence-indexed mutants are not available or are not
useful for specific studies (for example, in the case of
lethal mutations).
Deleteagene. Experimental approaches for identifying
DNA deletions in pools of mutants that are generated
by high-energy ionizing radiation have also been devel-
oped
23
. Although not yet widely popular in A. thaliana,
Deleteagene has several appealing advantages. For
example, it can be applied to plants in which trans-
formation is inefficient; it might also provide a means
to simultaneously mutate (delete) tandem duplicated
genes
23,73
. Deleteagene uses PCR primers that flank the
gene(s) of interest and DNA from pools of mutagenized
plants as a template. The presence of an amplification
product that is smaller than that expected for the wild
type identifies a deletion mutation (FIG. 2b). In spite
of the potential advantages of this technology, its use
in A. thaliana has been limited
106,107
, possibly in light
of competition with other successful reverse genetic
approaches. In non-model species, however, the wide
use of Deleteagene is hindered by the prerequisite of
knowing the sequence of the genes of interest and the
need for a large infrastructure to generate big mutant
populations.
Insertional mutagenesis. Identifying a T-DNA or
transposon insertion in a gene of interest is even more
straightforward. Gene-specific primers and T-DNA-
specific or transposon-specific primers are used in PCR
amplification from a DNA template that is derived from
pools of mutant plants. The presence of specific PCR
products in a particular pool indicates that a T-DNA or
transposon has inserted in or near the gene of interest in
one of the plants (FIG. 2c). One drawback of this approach
is that PCR using such complex pooled samples often
yields a high background of nonspecific amplification
products. This problem can be overcome by introduc-
ing a hybridization step (Southern blotting with a gene-
specific label) to identify the true-positive pools. This is
by far the most widely used reverse genetic approach in
A. thaliana. It has enabled the identification of hundreds
of specific mutants since the first successful screens were
carried out more than 10 years ago
108
.
Insertional mutagenesis also has its limitations, such
as the predominance of loss-of-function alleles, the biased
distribution of insertions in the genome, the inability to
characterize lethal mutations, and the difficulty of gen-
erating populations that are large enough to reach com-
plete saturation of the genome (saturation mutagenesis)
109
.
The identification of mutations in a specific gene
requires the screening of large numbers of mutagenized
lines, necessitating the construction and assaying of large
numbers of plant pools. This requirement has prompted
the development of more sophisticated pooling strate-
gies that minimize the number of assays required, but
still allow the identification of an individual mutant line
in one-step or two-step screens
30,110
.
Systematic reverse genetics
A major drawback of reverse genetic methods is that the
screen has to be repeated for every gene. To overcome
this problem, alternative and more effective mutation
detection approaches have been developed, and in many
cases they have supplanted the individual gene-by-gene
methods. High-throughput identification of genome
insertion sites in T-DNA-mutagenized and transposon-
mutagenized plant populations can be achieved by taking
advantage of the known sequence of the inserted DNA
and the availability of a complete genome sequence.
Various PCR-based strategies have been adapted for
this purpose (including thermal asymmetrical interlaced
PCR (TAIL PCR)
48
and the adaptor ligation approach
30
).
So far, more than 360,000 insertion sites have been
mapped in the A. thaliana genome, covering ~90% of
the genes (see the SIGnAL Salk Institute Genomic
REVI EWS

Accession
A sample of a plant variety that
is collected at a specific
location and time. The terms
ecotype, wild type and
accession are not uniformly
used in the Arabidopsis field
and often cause confusion. The
term accession is probably the
most appropriate way to
describe the Arabidopsis
laboratory lines that are
collected initially from the wild.
Virus-induced gene silencing
Gene silencing that is triggered
by a viral vector that encodes a
dsRNA with high sequence
identity to an endogenous
gene.
Analysis Laboratory web site). However, owing to the
near-random nature of insertional mutagenesis and
the selection of the lines to be analysed, these approaches
are no longer cost effective and more gene-directed
strategies are now needed to identify mutations in the
remaining 10% of untagged genes.
One solution is to make further use of the existing
T-DNA-tagged and transposon-tagged collections of
mutants that contain hundreds of thousands of unchar-
acterized (unsequenced) insertion sites (a typical T-DNA
line contains on average 1.5 expressed insertions with
nearly 50% of the lines containing 2 or more T-DNAs).
By using gene-specific and T-DNA/transposon-specific
primers, a directed gene-by-gene approach (as described
above) could identify mutations in the missing genes.
However, the most useful catalogue of mutations in
all genes in a plant genome will probably depend on
use of a range of mutagenesis approaches. Zinc-finger
nucleases, TILLING or Deleteagene strategies can be
used to obtain permanent, heritable mutations, and
gene-silencing approaches can be used to knock down
the activity levels of selected genes. RNAi constructs that
target every Arabidopsis gene are being developed
111
and
could be used to disrupt gene expression in any genetic
background, including the ever-growing collection of
natural accessions
112
. This tool will not only facilitate the
analysis of genes that have not been disrupted by inser-
tional mutagenesis methods (including essential genes
that can not be properly studied in the homozygous
knockout T-DNA/transposon lines owing to lethality)
but will also generate allelic series that represent different
levels of silencing.
Forward genetics with reverse genetic tools
One of the most exciting uses of the near complete col-
lection of gene-indexed A. thaliana mutations is the abil-
ity to carry out whole-genome forward genetic screens
113
.
Unlike classical forward genetic approaches, phenotypic
screening of gene-indexed collections can directly iden-
tify all the genes with knockout phenotypes that are
related to the process under investigation. Therefore,
dozens of genes that are involved in the process of interest
can be rapidly identified
16,106109
.
Two technical limitations currently stand in the way
of implementing genome-wide systematic screening
in A. thaliana. First, one (or ideally two) homozygous
mutations per gene need to be selected from the more
than 370,000 hemizygous sequenced mutants available.
Two are preferable because of the possibility that there
might be multiple mutations per plant, owing not only
to additional insertions but also to frequent deletions
and chromosomal rearrangements
114
, which prevent
direct genephenotype association without obtaining
further genetic confirmation. A set of homozygous
mutants that cover 25,000 Arabidopsis genes is cur-
rently being developed (see the Arabidopsis 2010 web
page). Second, systematic screening of tens of thou-
sands of individual mutant seed lines under many
experimental conditions will require the development
of seed-handling instrumentation that is not currently
available (FIG. 3).
Equally important to overcoming these two limita-
tions is the development of alternatives to stable inser-
tion and collections of point or deletion mutants in fixed
genetic backgrounds. Large collections of constructs
for use in RNAi and VIGS (virus-induced gene silencing)-
based techniques are also being developed
111
. When
applied in reverse genetic screens, as has been done in
D. melanogaster and Caenorhabditis elegans, and more
recently in human cells, these approaches have proved
effective in global gene-function studies
113
. For imple-
mentation in plants, several independent RNAi lines
per gene need to be obtained and characterized before
systematic gene analysis is carried out. For these types of
global study, VIGS (a transient assay) is an attractive and
rapid alternative, because the generation of stable trans-
formants can be avoided
115
; in principle, it can therefore
be applied to non-model organisms in which transfor-
mation is difficult or time-consuming
116
. However, cur-
rent VIGS systems can produce undesirable phenotypic
effects that are an unavoidable consequence of the viral
infection itself. Nonetheless, continued improvement of
this technology, along with the development of high-
throughput infection procedures, should allow this
approach to be adapted to genome-wide phenotypic
screens in various plant species.
The functional characterization of all genes in a
genome represents a formidable task that will require
the use of multiple methodologies in parallel. Typical
functional genomic approaches such as the use of
microarrays provide the desired high throughput, but
only limited functional information, whereas classical
genetic approaches are not suitable for genome-wide
studies. Recent advances in sequence-based mutant
detection have allowed for the generation of large
gene-indexed mutant collections and the concomi-
tant popularization of reverse genetic approaches.
Recombination-based cloning is facilitating the crea-
tion of large collections of vectors that are suitable
for RNAi, VIGS and the use of amiRNAs. In combi-
nation, these tools are allowing the functional char-
acterization of individual genes of interest in many
laboratories.
Several important advances towards gene-function
analysis in A. thaliana are on the horizon: the abil-
ity to do systematic forward genetics using reverse
genetic tools (simultaneous phenotypic analysis of
all gene-indexed mutants), the development of new
phenomic platforms, improvements in targeted
mutagenesis (specifically, homologous recombina-
tion), and the utilization of natural variation in gene-
function studies. Mutants or vectors that cover all of
the genes in the genome might become available in
2 or 3 years. It is more difficult to predict whether the
advances required for the systematic screening of these
mutants will be generated and/or how widely accessi-
ble they will become. New platforms to examine not
only morphological but also molecular phenotypes are
being developed
117,118
. The goal of these new phenomic
approaches is to enable systematic characterization of
gene-indexed mutants, yielding detailed information
about gene function. On the other hand, although
REVI EWS

List of gene-indexed mutants that show
resistance to compound Y
List of gene-indexed mutants that show
resistance to compound X
Plate 1
Plate 521
Plate 1
Plate 521
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 101112
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 1011
Plate
1
1
5
5
13
Position
A3
A4
F7
F8
D9
Gene name
At1g10540
At1g10540
At1g36710
At1g36710
At2g20010
Allele
1
2
1
2
1
Plate
1
1
8
8
23
23
Allele
1
2
1
2
1
2
Gene name
At1g20090
At1g20090
At1g49000
At1g49000
At3g00110
At3g00110
Position
D3
D4
A11
A12
H1
H2

Plate 521 of 521

12
2
At2g20010
D10
13
Plate 1 of 521 a b Screen 521 plates for resistance to compound X c Screen 521 plates for resistance to compound Y
Sequencing by synthesis
A group of new sequencing
technologies in which the
identity of the bases is
determined as they are added
to a newly synthesized DNA
molecule.
Ultra high-throughput
sequencing
A compendium of new
sequencing technologies that
have a common final aim of
accelerating (from years to
days or hours) and reducing
the cost (from millions to
hundreds or thousands of
dollars) of genome sequencing.
recent reports of increased efficiency of homologous
recombination in plants are promising, and the system-
atic use of this technology in functional analysis might
be just around the corner, further improvements (such
as high-throughput generation of recombination-ready
genomic constructs) will be required to enable the use
of this approach in genome-wide studies.
Induced mutations (point mutations, deletions, and
transposon- and T-DNA-generated mutations) and other
means of reducing gene expression (such as the use of
siRNAs, amiRNAs and artificial repressor proteins)
will continue to be used for some time. However, the
importance of natural allelic variation to study gene
function in plants is likely to increase. Paramount to
this shift from induced variation to natural variation
is that new technologies are needed for the rapid iden-
tification of such alleles. In this regard, sequencing by
synthesis and other new ultra high-throughput sequencing
(UHTS) technologies are expected to allow 1 gigabase
(78 coverage of the A. thaliana genome) of
sequence in 48 hours for a cost of US$3,000 (REF. 119).
The rapid advancement of UHTS is likely to have a
profound effect in two areas of gene-function analysis
in A. thaliana. First, induced alleles with phenotypes
that have only been observed in a specific genetic
background
120
point to the need for the creation and
sequencing of insertions in large mutant populations
using various accessions or ecotypes; a process that
will be facilitated by UHTS. Second, UHTS technol-
ogy will allow complete genome resequencing of many
hundreds, or even thousands, of accessions. With the
concomitant development of more phenotyping plat-
forms and corresponding community phenotyping
databases, whole-genome association studies that link
genotype and phenotype will become the approach
of choice to interrogate plant gene function and the
role of natural allelic variation in plant adaptation to
a range of local growth habitats
121
.
Figure 3 | Systematic reverse genetics. The next immediate goal in reverse genetics is to enable researchers to
test simultaneously the role of all the genes in the genome for involvement in a particular biological process. The
first step towards this goal involves generating non-redundant collections of homozygous mutants. From more
than 300,000 gene-indexed mutant lines in Arabidopsis thaliana, ideally two independent lines per gene
need to be selected, the mutations need to be confirmed and homozygous plants obtained. Panel a shows the
hypothetical end product of this step a collection of 521 96-well plates that correspond to 50,000 mutant lines,
two lines for each of the 25,000 genes. This seed library could then be systematically screened to study the role of
each one of the 25,000 represented genes in any given biological process. Panels b and c depict hypothetical
screens for genes that are involved in the response to compounds X and Y. The identification of the mutants
affected in the selected biological process (bigger plants in the compound X screen and viable plants in the
compound Y assay) allows the immediate identification of the underlying genes. By having two independent
mutant lines per gene, false positives and the need for experimental replicates are substantially reduced. In the
example of resistance to compound X, the growth of lines on plate 1, positions A3 and A4, strongly implicates
the corresponding gene At1g10540 in the response to the compound.
REVI EWS

1. Arabidopsis Genome Initiative. Analysis of the genome
sequence of the flowering plant Arabidopsis thaliana.
Nature 408, 796815 (2000).
2. Yamada, K. et al. Empirical analysis of transcriptional
activity in the Arabidopsis genome. Science 302,
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Acknowledgements
We thank A. Stepanova for comments on the manuscript.
J.R.E is supported by grants from the US National Science
Foundation, National Institutes of Health and Department of
Energy. J.M.A. is supported by North Carolina State
Uni versi ty and grants f rom the Nati onal Sci ence
Foundation.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
AHK2 | AHK3 | CKI1 | CRE1
FURTHER INFORMATION
Arabidopsis Biological Resource Center Stocks: http://
www.arabidopsis.org/abrc
Arabidopsis 2010: http://signal.salk.edu/gabout.html
Lehle Seeds: http://www.arabidopsis.com
New Wisconsin T-DNA Lines pDsLox Vector web site:
http://www.hort.wisc.edu/krysan/DS%2DLox
Nottingham Arabidopsis Stock Center:
http://arabidopsis.info
SIGnAL Salk Institute Genomic Analysis Laboratory web
site: http://signal.salk.edu
The Arabidopsis Information Resource homepage:
http://www.arabidopsis.org
Access to this links box is available online.
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After completion of the sequencing of the Arabidopsis

thaliana genome in 2000 (REF. 1), the plant biology

The Salk Institute for

Abstract | Genome sequencing, in combination with various computational and

Box 1 | Forward versus reverse genetic approaches

Possibility of generating allelic series, allowing the study of

Similar to gene silencing

Allows the identification of loss-of-function alleles, hypomorphs

Each of these approaches was used as a tool

Plate 521 of 521

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