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Functional markers in plants

Jeppe R. Andersen and Thomas Lubberstedt
Department of Plant Biology, Danish Institute of Agricultural Sciences, Research Center Flakkebjerg, 4200 Slagelse, Denmark

Different approaches (including association studies)

have recently been adopted for the functional characterization of allelic variation in plants and to identify
sequence motifs affecting phenotypic variation. We propose the term functional markers for DNA markers
derived from such functionally characterized sequence
motifs. Functional markers are superior to random DNA
markers such as RFLPs, SSRs and AFLPs owing to complete linkage with trait locus alleles. We outline the
definition, development, application and prospects of
functional markers.
Genetic markers were originally used in genetic mapping
to determine the order of genes along chromosomes. In
1913, Alfred H. Sturtevant generated the first genetic map
using six morphological traits (termed factors) in the fruit
fly (Drosophila melanogaster) [1] and, soon after, Karl Sax
produced evidence for genetic linkage between a qualitative and a QUANTITATIVE TRAIT LOCUS (see Glossary) (seed
color and seed size) in the common bean (Phaseolus
vulgaris) [2]. Since these pioneer studies, genetic markers
have evolved from morphological markers through isozyme markers to DNA markers. Today, genetic markers
are used in both basic plant research and plant breeding to
characterize plant germ plasm, for gene isolation, for
marker-assisted introgression of favorable alleles and for
variety protection [3].
Morphological markers are easily monitored but can be
affected by the environment. They are limited in number
and some (e.g. flower color) appear late in plant development, making early scoring impossible. In addition, a
given morphological marker can affect other morphological markers or traits of interest in breeding programs
because of PLEIOTROPIC gene action. Finally, the usefulness of morphological markers is restricted by their
limited number. Consequently, complete genome assays,
as required for quantitative trait locus (QTL) analyses, for
example, are not feasible for morphological markers.
Similar limitations apply to the application of isozyme
markers [4].
With the advent of DNA markers [5,6], these limitations
have been overcome. A DNA marker is typically derived
from a small region of DNA that shows sequence
polymorphism between individuals within a species.
Thousands of phenotypically neutral, random DNA
markers (RDMs) can be generated for any species and
have been successfully used in many studies to represent
genomes in biodiversity studies [7] or to map trait
genes (http://www.zmdb.iastate.edu/PlantGDB/). However,

genetic linkage between a specific RDM and a target locus

allele, established by QTL studies, for example, can be
broken by genetic recombination; this limits the use of
RDMs as a diagnostic tool [8]. Recently, projects on
structural and functional genomics have been established
for several crop species (e.g. http://www.grasp-euv.dk/;
http://www.maizegenetics.net/). The knowledge generated
in these projects will allow systematic development of
functional markers (FMs), which are derived from polymorphic sites within genes causally affecting phenotypic
trait variation.
So far, DNA markers have been compared primarily on
their technical properties [6]. By contrast, RDMs, gene
targeted markers (GTMs) and FMs are here defined based
on the level of functional characterization of the polymorphisms monitored by these marker types (Figure 1,
Table 1). RDMs are derived at random from polymorphic
sites in the genome, whereas GTMs are markers derived
from polymorphisms within genes. Both RDMs and GTMs
can be developed independently of their relationship to
any phenotypic characters. By contrast, FMs are derived
from polymorphic sites within genes causally involved in
Glossary
Allogamy: cross fertilization.
Association studies: linkage disequilibrium mapping; a method based on
linkage disequilibrium in populations for identifying particular alleles
associated with a given phenotype.
Autogamy: self fertilization.
Ecovalence: measure of phenotypic stability of a given genotype.
Foreground selection: selection by genetic markers to ascertain the presence
of an introgressed gene when direct phenotypic evaluation is not feasible.
Genetic drift: the random change of the occurrence of a particular gene in a
population, genetic drift is thought to be one cause of speciation when a group
of organisms is separated from its parent population.
Genetic marker: polymorphic genetic property that can be used to distinguish
the parental origin of alleles; markers originate from DNA sequence
polymorphisms.
Isogenic lines: a pair of isogenic lines that is genetically identical except for
specified sequence motifs.
Linkage disequilibrium: the non-random occurrence of alleles in haplotypes;
when the observed frequencies of haplotypes in a population does not agree
with haplotype frequencies predicted by multiplying together the frequency of
individual genetic markers in each haplotype; linkage disequilibrium can be
caused by selective constraints or the close proximity of two or more loci on
the chromosome.
Linkage drag: the reduction in fitness in a cultivar caused by deleterious genes
introduced along with a beneficial gene (e.g., during backcrossing); the
presence of individual genes can be monitored using DNA markers, and the
risk of linkage drag thus reduced.
Marker haplotype: a set of closely linked genetic markers present on one
chromosome that tend to be inherited together (not easily separable by
recombination); some haplotypes might be in linkage disequilibrium.
Pleiotropy: multiple effects of a single gene that affects more than one
phenotypic character.
Quantitative trait locus: the location of a gene that affects a trait that is
measured on a quantitative scale; these traits are typically affected by more
than one gene as well as the environment.

Corresponding author: Thomas Lubberstedt (Thomas.Luebberstedt@agrsci.dk).

http://plants.trends.com 1360-1385/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2003.09.010

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phenotypic trait variation. DNA markers derived from

functionally defined sequences are mentioned in the
context of plant breeding [9 11] and biodiversity studies
[12], as well as in human genetics [13,14]. Terms such as
functional, gene targeted and diagnostic markers have
been used [10 12] but have not been clearly defined. Our
major objectives in this article are: (i) to define FMs and
to distinguish them from other marker types; (ii) to outline FM development in plants; and (iii) to discuss the
prospects for and limitations of FM application in plants.
Development and validation of functional markers
Functional marker development requires allele sequences
of functionally characterized genes from which polymorphic, functional motifs affecting plant phenotype can
be identified. Furthermore, the use of FMs depends on the
availability of robust marker assay technologies, described
elsewhere [6].
Functionally characterized genes
The starting point of FM development is the sequence of a
gene with an assigned function. Accumulation of plant
nucleotide sequences proceeds exponentially, with more
than 24 million entries deposited at GenBank (April 2003;
ftp://ftp.ncbi.nih.gov/genbank/gbrel.txt). In the model
plant Arabidopsis, fewer than 10% of its , 25 000 genes
have been functionally characterized [15]. In other species,
the number of functionally characterized sequences is
substantially lower. However, based on sequence homology, putative functions can be assigned to 30 50% of the
expressed sequences in any species [16]. This candidate
gene approach and synteny relationships between plant
genomes [17 19] have been successfully exploited to
identify agronomically relevant genes [20,21]. In addition,
high-throughput assays, such as expression profiling,
have been developed recently [15] to identify candidate
genes on a large scale. Other methods, including RNA
interference [22], T-DNA and transposon gene tagging
[23,24] and gene expression QTL mapping [25] can be used
to determine gene function. In conclusion, candidate genes
can be identified for most agronomic traits and crop species
(Box 1) and GTMs can be derived (Figure 1).
Indirect proof of allele function
Polymorphic allele sequences are a prerequisite for FM
development. So far, the focus has been on massive genome
or expressed sequence tag (EST) sequencing of one or a few
genotypes per species [26 28], whereas comprehensive
allele sequencing has been conducted for only a few
genes [29,30]. Nucleotide diversity determined as average

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sequence divergence between two individuals at a given

locus varies considerably between [31,32] as well as within
[33 37] species, and is generally lower in AUTOGAMOUS
than in ALLOGAMOUS species [38,39]. The challenge of FM
development is to associate sequence polymorphisms with
phenotypic variation.
ASSOCIATION STUDIES , originally established in human
genetics [40], aim to identify genes and even functional
motifs within genes that affect phenotypic characters
[29,30]. This approach relies on LINKAGE DISEQUILIBRIUM
(LD) mapping based on nonrandom occurrence of allele
haplotypes in the genome [41]. In addition to comprehensive allele sequencing, the availability of phenotypically
characterized plant populations is crucial for association
studies. Distinguishing the effects of different intragenic
polymorphisms on phenotypic variation requires high
genetic resolution (i.e. low LD levels). In Arabidopsis, LD
decays over tens of kb [42], whereas, in maize, LD generally decays within 1 kb [29,43]. Thus, in maize and other
species with similarly low levels of LD, association studies
have the potential to identify sequence motifs, such as a
few nucleotides or insertions/deletions, that affect trait
expression. Furthermore, association studies can provide
the allele effects of such motifs. However, association
studies might not be sufficient to distinguish causative
from phenotypically neutral polymorphisms in extended
haplotype structures [29]. Furthermore, the genetic background might affect results from association studies, and
statistical approaches have been developed to control
unknown population structures [44]. Because association
studies provide only indirect (statistical) evidence of
sequence motif function, we suggest the term indirect
FMs (IFMs) (Figure 1) for FMs derived by this method.
Direct proof of allele function
Direct proof of sequence motif function can be obtained by
comparing isogenic genotypes differing in single sequence
motifs. Isogenic genotypes can be produced by: (i) induced
mutation followed by screening of large populations by
targeting-induced local lesions in genomes (TILLING);
and (ii) homologous recombination (HR).
TILLING was developed in Arabidopsis for efficient
screening of ethylmethane sulfonate (EMS) induced
mutations, which produce subtle mutations, primarily
C/G to T/A transitions. Consequently, an allelic series of
mis-sense mutations can be obtained for each target gene
[45]. Applying this method in the legume Lotus japonicus,
one domain-specific mutation per 3000 plants was detected
in the SYMRK gene [46]. Thus, combined with phenotyping, TILLING provides direct proof of function of both

Table 1. Comparison of marker types

Marker type

Origin of DNA
sequence

Function of
polymorphic site

Method for functional

sequence motif
characterization

Marker
development
costs

Quality of
markera

RDM
GTM
IFM
DFM

Unknown
Gene
Gene
Gene

Not known
Not known
Functional motif
Functional motif

Association studies
Isogenic lines

Low
Low
Medium
High

Low
Medium
High
High

Abbreviations: DFM, direct functional marker; GTM, gene-targeted marker; IFM, indirect functional marker; RDM, random DNA marker.
a
Based on risk of recombination and functional characterization of marker.
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Box 1. Examples of candidate genes for functional marker development

One example of a functionally characterized gene is Dwarf8 in maize [64]
(Table I), which encodes a gibberellin response modulator from which
functional markers (FMs) can be developed for plant height and
flowering time. Orthologs to Dwarf8 have been identified in Triticum
aestivum (wheat; Rht1) [64], Oryza sativa (rice; SLR1) [65] and Hordeum
vulgare (barley; sln1) [66], and were involved in the development of
high-yielding wheat and rice varieties during the Green Revolution
[74]. Altered function of alleles in these orthologous genes [64 66] can
reduce the response to gibberellin and consequently lead to decreased
plant height. Thus, biallelic (gibberellin sensitive/insensitive) FMs can
be derived for targeted and rapid development of varieties with
increased lodging tolerance.
Association studies have been used to relate individual functional
motifs within the Dwarf8 wild-type allele with variation in flowering time
among maize inbreds [29]. Functional motifs can be identified in DNA
regions controlling gene expression (enhancer or promoter), mRNA
stability, protein expression and stability, or DNA regions specifying

amino acid residues affecting (for example) enzymatic activity. In

Dwarf8, nine polymorphisms (located in both transcribed and 50 untranscribed regions of the gene) showed significant association, one
particular 6 bp deletion accounting for a flowering time difference of 7
11 days between inbreds. In this study, individual polymorphisms were
biallelic. These biallelic FMs could be combined yielding multiallelic FM
haplotypes. Theoretically, 29 (i.e. 512) haplotype markers could be
derived from this study, but fewer haplotype FMs would be sufficient for
fine-tuning of flowering time. Such haplotype FMs could be developed
both within and between genes, depending on the trait of interest.
Dwarf8 is an example of a pleiotropic gene (a gene that affects more
than one phenotypic character). Therefore, selecting this allele for
decreased plant height would conflict with the use of Dwarf8 for
flowering time control. Consequently, flowering time control would
require FMs derived from additional flowering time genes, in addition
to using Dwarf8-derived FMs. Thus, pleiotropic effects constitute a
potential problem and need to be determined before application of FMs.

Table I. Candidate genes for functional marker development

Trait

Gene(s)

Species

Refs

Plant height
Plant stature
Flowering time
Vernalization requirements
Fruit size
Food quality
Feed quality
Disease resistance
General stress response

Dwarf8 orthologs
tb1
Dwarf8
VFR2
fw2.2
GBSS
Genes in the lignin biosynthesis pathway
NBS LRR class of resistance genes
ERF transcription factors

Cereals
Maize (Zea mays)
Maize (Zea mays)
Oilseed (Brassica rapa)
Tomato (Lycopersicon ssp.)
Rice (Oryza sativa)
Several
Several
Several

[64 66]
[67]
[29]
[68]
[69]
[70]
[71]
[72]
[73]

Abbreviations: ERF, ethylene-responsive element binding factor; NBSLRR, nucleotide binding site leucine-rich repeat.

induced and natural polymorphisms. The method is

currently being transferred to crop plants (http://www.
evry.inra.fr/public/projects/tilling/tilling.html). Because
EMS mutagenesis introduces many mutations, backcrossing to the original wild type might be required before
phenotypic analysis.
Alternatively, transformation of allelic series into
isogenic genetic backgrounds can confirm the function of
individual sequence motifs. However, current plant transformation protocols based on nonhomologous end joining
result in random genomic integration of transgenic DNA,
position effects, multiple insertions of the transgene and
transgene alterations [47], obscuring quantitative phenotypic differences between alleles. This can be circumvented using HR-based, locus-targeted integration of
alleles [47]. HR has proved to be difficult in higher plants
[48] but has been established in the moss Physcomitrella
patens, in which nine out of ten integration events were
shown to be a result of HR [49]. Recently, , 1% of insertion
events in rice (Oryza sativa) were found to result from HR
[50]. In conclusion, although HR in crop plants might
become available soon, EMS mutation/TILLING currently
seems to be the most applicable method for providing
function of single DNA sequence motifs. We suggest
terming FMs derived from such functionally proven motifs
direct FMs (DFMs) (Figure 1).
Comparison of marker types
FMs are superior to GTMs and RDMs in several applications owing to their complete linkage to functional
http://plants.trends.com

motifs (Table 1). In contrast to RDMs or GTMs, FMs allow

reliable application of markers in populations without
prior mapping, the use of markers in mapped populations
without risk of information loss owing to recombination
and better representation of genetic variation in natural
or breeding populations. More generally, FMs are useful
for: (i) more efficient fixation of alleles in populations;
(ii) controlled balancing selection; (iii) screening for alleles
in natural as well as breeding populations; (iv) combination of FM alleles affecting identical or different traits
in plant breeding; and (v) construction of linked FM
haplotypes.
Using markers in populations without prior mapping
The predictive values of RDMs and GTMs depend on the
known linkage phase between markers and target locus
alleles. In an RDM-based QTL study of different forage
traits, four maize populations [A B-l (large population),
A B, A C, C D (small populations)] were compared
that were derived from crosses of four flint inbreds [51].
Comparing the A B-l and A B populations, 60% of the
QTLs were shared, whereas only 38% and 30% were
shared when comparing A B-l with A C, and A B-l
with C D, respectively. Furthermore, the selection of
some RDM alleles that were favorable in cross A B would
have been detrimental in cross CxD [51]. Thus, RDMbased QTL mapping is necessary for each cross de novo,
because different subsets of QTLs are polymorphic in
individual populations (non-detection of expected QTL)
and linkage phases between RDM and QTL alleles can

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Level of functional characterization

of polymorphisms monitored by
markers

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Vol.8 No.11 November 2003

Number of alleles
sequenced per
marker gene

Methods for marker

development

No
alleles

Restriction enzymebased methods

Marker type

RDM

Sequencing
One
allele

Expression profiling,
sequence comparisons,
synteny studies

GTM

Sequencing
At least
two
alleles

Association studies

IFM

Mutant studies,
EMS/TILLING,
HR

DFM

TRENDS in Plant Science

Figure 1. Definition of marker types based on the level of functional characterization. Increasing thickness of arrow indicates increasing functional characterization of polymorphisms monitored by markers. Random DNA markers (RDM), gene-targeted markers (GTM) and functional markers (FM) are defined based on this level of functional
characterization. RDMs are derived at random from polymorphic sites in the genome whereas GTMs are markers derived from polymorphism within genes. Both RDMs
and GTMs can be established independently of the relationship of the underlying sequence polymorphisms to phenotypic characters. By contrast, FMs are derived from
polymorphic sites within genes causally involved in phenotypic trait variation. Indirect and direct FMs (IFM) and DFM) differ in development procedures and thus in the
degree of the functional characterization of polymorphisms. The list of methods for marker development is not comprehensive. Abbreviations: EMS, ethylmethane sulfonate; HR, homologous recombination; TILLING, targeting-induced local lesions in genomes.

disagree even in closely related parent genotypes of

mapping populations.
By contrast, once genetic effects have been assigned to
functional sequence motifs, FMs derived from such motifs
can be used to fix gene alleles (defined by one or several FM
alleles) in several genetic backgrounds without additional
calibration. This would be a major advance in marker
applications, particularly in plant breeding, to select (for
example) parent materials to build segregating populations, as well as subsequent selection of lines (line
breeding) or inbreds (hybrid breeding). Depending on the
mode of FM characterization, FMs can also be used for
targeted combination of alleles in hybrid and synthetic
breeding. In population breeding and recurrent selection
programs, FMs can be used to avoid GENETIC DRIFT at
characterized loci. In addition, FMs can be used in variety
testing based on the presence or absence of specific alleles
at morphological trait loci currently used to discriminate
varieties.
Application of markers in mapped populations
The usefulness of RDMs (or GTMs) within the same cross
as in which marker-trait linkages have been established
depends on the persistence of linkage between RDM and
target locus alleles. This is a function of genetic distance
between RDM and target locus [52], number of genes to be
traced [53] and number of generations since linkage was
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established [54]. Consequently, phenotypic validation is

required at least once in RDM or GTM assisted selection to
confirm the presence of the target gene(s). This validation
can be circumvented by using FMs for (for example)
FOREGROUND SELECTION [55] in backcrossing programs.
Owing to the scarcity of recombination events, large
chromosome segments are transmitted between generations. Consequently, undesirable genes might be linked to
the target gene, particularly if non-adapted genotypes
served as donor [56]. Because FMs are derived directly
from functional motifs and thus are in complete linkage
with the favorable target locus allele, foreground selection
based on FMs would be more efficient than RDM-based
foreground selection.
In grasses, it has been shown that genes are typically
clustered in recombinationally active gene-rich islands of
the genome separated by blocks of high-copy DNA [57,58].
In the longer term, it would be desirable to develop FMs for
all genes linked in such islands and to characterize FM
haplotypes with respect to agronomic performance.
Representation of genetic variation in natural and
breeding populations
The number of alleles for a gene of interest might disagree
with the number of alleles of RDMs (or GTMs) genetically
linked to it. For example, (biallelic) amplified fragment
length polymorphism (AFLP) marker loci are scored for

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the presence or absence of a given marker band. In

AFLP-based biodiversity studies, all genotypes classified into the same marker class (e.g. all carriers of a
given AFLP marker band) are usually considered to be
identical for this marker locus. However, the allelic
composition of chromosome regions linked with a given
AFLP marker allele might be quite diverse. Thus,
(combinations of) FMs can more accurately describe the
actual diversity of relevant genes in plant breeding or
biodiversity studies [12], and can be used to screen for
or to record known alleles in natural and breeding
populations. However, if FMs have not been developed
directly for sequence motifs that exhibit ecological or
agronomical relevant variation, multiallelic RDMs
(e.g. simple sequence repeats), might equally well describe
allelic diversity for such genes. Furthermore, knowledge is
generated during FM development about the nature and
physical location of sequence motifs affecting phenotypic
expression, which can subsequently be applied to targeted
search or development [59] of alleles in plant germ plasm.
Prospects for and limitations of functional markers
Several target genes for FM development are available
(Box 1) and allele sequencing projects are in process
in Arabidopsis (http://walnut.usc.edu/) as well as in
crop species (e.g. http://www.grasp-euv.dk/; http://www.
maizegenetics.net/). However, even in model species, only
, 10% of all genes have been functionally characterized,
whereas (for non-model species) this number is probably
less than 5%. Furthermore, the criteria by which such
genes have been functionally characterized (e.g. in a
biological sense) might not be sufficient to establish gene
function in an agronomic sense. Thus, further functional
characterization might be necessary before proceeding to
FM development. Furthermore, a crucial question will be
whether useful allelic variation can be identified for all
genes of ecological and agronomical relevance. Given that
functionally described target genes are available, the
limiting step in FM development will be the choice and
development of suitable plant materials and its thorough
phenotypic characterization to distinguish minor phenotypic effects.
IFMs are derived by association studies in large
genotype collections, allowing for generalization of estimated gene effects of markers. DFMs are derived by
comparing ISOGENIC LINE pairs, for which development
costs are high, limiting initial investigations to isogenic
comparisons in one or few genetic backgrounds. To reduce
development costs, association studies could be applied to
select candidate sequence motifs (IFMs) for further testing
in isogenic comparisons yielding DFMs. In the longer
term, DFMs need to be evaluated in different genetic
backgrounds to obtain more precise estimates of gene
effects. Furthermore, the phenotypic stability of FMs
needs to be adequately described, comparable to ECOVALENCE at the genotype level [60]. Thus, refining the
predictive value of FMs will require the development of an
experimental, statistical and bioinformatic framework for
data accumulation and evaluation across different studies.
Association studies and isogenic comparisons of homozygous genotypes can be extended to test crosses using
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Vol.8 No.11 November 2003

different testers to determine both additive and dominance

effects of functional sequence motifs. Estimates of additive
and dominance effects are determined in a relative manner
(i.e. the estimates depend on the trait locus alleles as well
as the genetic background of genotypes present in a
specific study). Thus, to integrate data across experiments,
common reference genotypes need to be used in all studies.
Both isogenic line development and association studies
are facilitated by the ability to produce inbreds. In species
with self-incompatibility mechanisms or a high degree of
inbreeding depression, heterogeneous populations need to
be compared that are fixed for different FM alleles. Thus,
the identification of small FM effects will be complicated by
genetic variation within test units. Consequently, FM
development is more feasible for autogamous or selfcompatible and inbreeding-tolerant allogamous species
than for strictly allogamous species. However, autogamous
crops typically have extended haplotype structures. In
these species, either isogenic comparisons need to be used
to assign function to sequence motifs or highly recombined
populations need to be generated before association
studies.
Application of marker-assisted selection (MAS) has
been shown to increase selection efficiency, particularly for
traits with low heritability [61]. However, selecting for
many genes with small effects has been shown to decrease
the efficiency of MAS [62]. Therefore, it is important to
consider the optimal number of genes to be involved in FM
development for quantitative traits. Using many key
genes ensures a better control of the trait and provides
flexibility in case of pleiotropic effects of specific genes.
However, using fewer FMs will substantially reduce
development costs and reduce the risk of LINKAGE DRAG
of undesirable alleles. Furthermore, QTL studies have
revealed skewed distributions of QTL effects, with few
QTLs having large effects and several having moderate or
small effects on a given trait [63]. Taking these factors and
the laborious nature of FM development into account,
initial FM development should concentrate on genes that
confer substantial phenotypic variation. Thus, selection of
relevant key genes will be a crucial and limiting step in
FM development.
Acknowledgements
We thank Torben Asp, Ursula K. Frei, Christina Ingvardsen, Louise
B. Jensen and two anonymous reviewers for valuable comments during
preparation of the manuscript. Financial support came from the Danish
Ministry of Food, Agriculture and Fisheries.

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