330 Opinion TRENDS in Plant Science
7 July 2003
Breeding by Design
Johan D. Peleman and Jeroen Rouppe van der Voort
Keygene N.V. Business Unit Genetics, PO Box 216, 6700 AE Wageningen, The Netherlands
Breeding by Designe is a concept that aims to control
all allelic variation for all genes of agronomic import-
ance. This concept can be achieved through a combi-
nation of precise genetic mapping, high-resolution
chromosome haplotyping and extensive phenotyping.
Thanks to marker technology, software tools and the
know-how available today, this goal can now be
achieved. Depending on the crop-specific generation
time, controlled marker-assisted selection strategies
could lead to the production of superior varieties within
five to ten years.
The potential value of genetic markers, linkage maps and
indirect selection in plant breeding has been known for
more than 80 years. However, it was not until the advent of
DNA marker technology in the 1980s, that a large enough
number of environmentally insensitive genetic markers
could be generated to follow the inheritance of important
agronomic traits adequately. Since then, DNA marker
technology has dramatically enhanced the efficiency of
plant breeding. In the past decade, many breeding com-
panies and research institutes have, to varying degrees,
started using molecular markers to increase the effective-
ness of selection in breeding and to shorten the develop-
ment time of varieties significantly [1,2]. Currently,
advances in automated technology have led to the deve-
lopment of a new approach in marker-assisted breeding.
The advances in applied genomics and the possibility of
generating large-scale marker data sets provide us with
the tools to determine the genetic basis for all traits of
agronomic importance. In addition, methods for assessing
the allelic variation at these agronomically important loci
are now available. This combined knowledge will even-
tually allow the breeder to combine the most favourable
alleles at all these loci in a controlled manner, leading to
superior varieties.
The ‘Breeding by Design’ strategy is an extension of the
different levels of marker applications that are currently
used in breeding. These levels will be discussed briefly
before addressing the Breeding by Design concept in more
detail.
Marker-assisted selection
Marker-assisted selection (MAS) using DNA markers
instead of phenotypic assays reduces the cost and
increases the precision and efficiency of subsequent selec-
tion steps applied in breeding [1– 3]. A successful example
of this strategy is marker-assisted backcrossing (MABC),
which is applied in a large variety of crops. In MABC,
Corresponding author: Johan D. Peleman ( johan.peleman@keygene.com).
http://plants.trends.com 1360-1385/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00134-1
Vol.8 No.
mapped markers are used to select backcross progeny with
the highest percentages of recurrent parent genome
together with a minimum number of donor segments.
MABC programs reduce the number of generations needed
for recovery of the recurrent genome from eight to three
in maize [4].
Marker-assisted breeding
Where breeding goals cannot be achieved using traditional
approaches, there is now considerable scope for using
molecular markers to develop new varieties. DNA markers
can add significantly more value than just improving
the speed, cost or quality of existing breeding programs.
Here, the limitation is not the available technology, but
rather the challenge facing the (molecular) breeder to
find creative approaches for developing new products. By
understanding the genetic basis of complex traits (or
obstacles), it becomes possible to control them. Several
examples of successful marker-assisted breeding (MAB)
approaches are now emerging: for example, a method for
PYRAMIDING (see Glossary) dominant resistance genes [5]
and the breeding of an aphid (Nasonovia ribisnigri)-
resistant lettuce variety by introducing a recessive resist-
ance gene surrounded by severe LINKAGE DRAG [6].
Breeding by Design
The examples above show that applying markers in
breeding not only improves existing selection processes
but can aid in creating novel varieties bearing new
characteristics of agronomic importance. Building on
these capabilities, by understanding the genetic basis of
all agronomically important characters and the allelic
variation at those loci, the breeder would be able to design
superior genotypes ‘in silico’. We have called this concept
‘Breeding by Designe’. This goal can be reached by
following a three-step approach.
(1) Mapping loci involved in all agronomically relevant
traits
To elucidate the genetic basis of agronomically important
traits, mapping populations are needed in which those
Glossary
Heterosis: Hybrid vigour.
Linkage drag: Co-inheritance of undesirable trait(s) with a gene of interest.
Typically, this is a phenomenon that can be observed during backcross
breeding using an exotic donor parent.
Pyramiding: The accumulation of several desirable traits in the same genotype
through backcross breeding.
 Opinion TRENDS in Plant Science
traits are segregating. To map all traits that are relevant
in breeding a crop, we prefer to use introgression line (IL)
libraries (Box 1). Considerable progress has been made in
constructing IL libraries for several different crops over
the past five years [7]. IL libraries are extremely powerful
tools in the quest to map the loci underlying all agron-
omically important traits. The key advantage of IL
libraries is in reducing the complexity of polygenic traits
by separating them into a set of monogenic loci (Box 1).
To identify the allelic variation at each locus of interest
by using ‘chromosome haplotyping’ (see further), it is
essential to determine a precise position for the loci of
Box 1: Mapping advantages of introgression line libraries
An introgression line (IL) library consists of a series of lines harbouring
a single homozygous donor segment introgressed into a uniform,
cultivated background (Fig. I) (reviewed in [7]). The advantages of IL
libraries compared with other mapping approaches are:
† IL libraries consist of homozygous ‘immortal’ lines and therefore can
be phenotyped repeatedly and used for the simultaneous mapping of
many traits.
† IL libraries contain homogenous genetic backgrounds, only differing
from one another by the introgressed donor segment. Thus, epistatic
effects from the donor parent are eliminated.
† Quantitative trait loci (QTL) are dissected into separate monogenic
components, which increases the reliability of measuring phenotypic
traits
† ILs containing interesting QTL can be backcrossed to various lines to
investigate interactive effects.
† Although dependent on the resolution of the IL library (i.e. the
average introgression segment size), QTLs are typically mapped into
smaller intervals than by classical QTL mapping.
† IL libraries provide optimal starting material for the fine-mapping of
the mapped loci and for generating improved breeding lines.
BC1
BC1 selection
BC2 selection
BC3 selection
BC3S1 selection
Fig. I. Example of an introgression line (IL) library construction process in tomato (12 chromosomes each separated by a grey bar) using Marker-Assisted BackCrossing.
Each horizontal bar represents an individual [best visible in the BC1 (BackCross generation 1) selection]. Homozygous recurrent parent segments are shown in red.
The homozygous donor segments are indicated in blue. Green bars represent heterozygous segments. The construction and use of IL libraries is extensively reviewed
in [7].
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Vol.8 No.7 July 2003 331
interest. IL libraries also provide perfect starting material
for this purpose: each line containing a locus of interest can
be backcrossed to the recurrent parent (and, if necessary,
selfed) to create a large segregating population. This
population can be used to identify recombinants within the
introgression segment using flanking markers. Phenotyp-
ing these recombinants enables the locus to be mapped at
high resolution.
Another powerful fine-mapping approach is provided by
the wealth of sequence information available from model
plant species combined with the rapidly expanding under-
standing of gene function. This knowledge can be linked to
† A secondary bonus of using IL libraries is that often new ‘exotic’
alleles can be found that have a positive effect in the culture crop
germplasm.
To study interaction (epistasis) between non-allelic genes,
whereby the expression of one gene interferes with the expression
of the other gene, reciprocal IL libraries can be constructed. In such a
case, IL libraries from line A into B and vice versa are constructed. By
doing so, phenotypes that cannot be detected because they are
mediated by interacting loci in the A £ B library will be measured as a
knocked-out phenotype in the B £ A library. Subsequently, crosses
between individual introgression lines each bearing one of the
interacting alleles can be made to investigate the extent of the
interaction [17].
To map loci contributing to heterosis, the IL library can be crossed to a
tester parent. This will create an F1 IL library in which each introgression
segment is present in the heterozygous state. This F1 IL library is then
phenotyped to detect heterotic effects caused by specific introgression
segments.
TRENDS in Plant Science
332 Opinion TRENDS in Plant Science
commercial species by exploiting synteny between both
species, thereby opening the possibility for developing
candidate-gene mapping approaches and causal SNP
markers [8,9]. In our view, the candidate gene approach
can provide a shortcut in the fine mapping or cloning of loci
that are roughly mapped using an IL library.
Linkage Disequilibrium Mapping (LD Mapping) pro-
vides an alternative but more risky approach to fine-map
loci of interest. LD mapping relies on associations between
the phenotype and neutral polymorphisms located near
the trait locus [10].
Because of the complexity of this approach and the
associated risks, our preference is to use ‘targeted LD
Mapping’. Once the approximate position of a locus is
known, LD mapping can be applied using markers (Box 2)
in the area of that locus, leading to the identification of
markers or haplotypes that show a strong association with
the phenotype under investigation, thereby fine mapping
the locus of interest [11].
(2) Assessment of the allelic variation at those loci
Although several strategies based on different population
structures exist to unravel the genetic basis of complex
traits, it is still impossible to predict the phenotypes
generated by those genes in the germplasm. This is
because of the allelic variation that exists in the germ-
plasm at those loci. Typically, in a segregating population
(F2, BackCross, recombinant inbred lines, double hap-
loids, IL library) only two alleles are segregating per locus.
Mapping the genes involved in a desired phenotype only
enables the prediction of the phenotype within the same
population or within populations in which the same alleles
are segregating at that locus. To obtain broad predictive
power, we need to be able to distinguish all alleles at the
locus of interest and to assign phenotypic values to the
different alleles.
As mentioned above, a potential short cut to mapping
genes and allelic variation simultaneously could be
achieved using association mapping with single markers
[10]. However, a better method for assessing allelic vari-
ation at loci of interest is by using marker haplotypes.
Using a set of tightly linked markers at a locus, the
combined scores of all markers in principle can distinguish
all the different alleles that occur at that locus in a set of
accessions (Box 2). For example, a high correlation was
found between resistance phenotypes and marker haplo-
types derived for the largest resistance gene cluster in
lettuce [12].
By extending this technique to the whole genome,
complete ‘chromosome haplotypes’ can be generated
(Fig. 1). Assuming a high level of saturation with markers,
these chromosome haplotypes enable us to determine the
allelic variation at any position in the genome. After fine
mapping the genes of agronomic importance, for example,
by using IL libraries combined with targeted LD mapping,
these chromosome haplotypes can be used to assess the
allelic variation at those loci.
Once the allelic variation at the loci of interest is
identified, it is essential to attribute phenotypic values to
the different alleles. For this purpose, the inbred lines
bearing different alleles at the loci of interest need to be
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Vol.8 No.7 July 2003
Box 2: Predicting trait values by use of haplotypes
The predictive value of a marker for a trait of interest in a broad
germplasm is determined by the mode of inheritance of the trait, the
recombination distance between the marker and the trait locus, and
the mutation rate of the marker. Instead of single-marker-based tests,
combinations of linked marker alleles at the same chromosome
(haplotypes) can be used to predict phenotypic trait values in a panel
of lines. Haplotypes of multiple markers have a higher information
content compared with single markers. Therefore, an increased
predictive power is achieved by using haplotypes. We have success-
fully applied this approach in different crop species by using AFLP
markers to identify and predict different alleles of agronomically
important loci.
Figure I shows an example of three allelic variants of a hypothetical
gene. Three tightly linked single nucleotide polymorphism (SNP)
markers were identified in a segregating population derived from
parental lines G1 and G2. However, once the linked markers are used
in a germplasm panel (G1 –G6), typically the association between
(most of) the markers is lost because of the independent origin of the
SNPs. In this example, the haplotypes composed of the three SNPs
are required to discriminate the three different alleles.
Haplotype-based analyses of SNPs are the focus of intensive
research because of the effectiveness of using haplotypes in
(fine) mapping of complex traits and scanning for allelic variants
(e.g. [18,19]).
SNP1 SNP2 SNP3
G1 – + +
G2 + – –
G3 + – –
G4 + + –
G5 + – +
G6 + + +
TRENDS in Plant Science
Fig. I. Predictive value of haplotypes. G1 and G2 represent parents of a segre-
gating population, G3–G6 represent germplasm lines. Four gene alleles are
distinguished: orange, white, green and blue. For clarity, the genotypes of
three SNP (single nucleotide polymorphism) markers are indicated as ‘ þ /2‘
scores. The white allele is indicative of two different haplotypes whereas the
orange, green and blue alleles correspond to unique haplotypes. In this
example, lines G3 and G4 have different haplotypes while bearing the same
gene allele owing to a point mutation at the SNP2 locus.
thoroughly phenotyped. In the case of polygenic traits, it
is possible to pre-select those lines that bear different
combinations of alleles at the different loci for phenotyp-
ing. Currently, we are developing algorithms that can
combine those data with the phenotypic data on the lines
to determine the contributing value of each allele to the
phenotype.
In our view, the combination of mapping loci using
simple mapping populations, in combination with allele
 Opinion TRENDS in Plant Science Vol.8 No.7 July 2003 333

1 2

TRENDS in Plant Science

Fig. 1. Chromosome haplotypes. Chromosome haplotypes of a set of maize lines for one chromosome. Horizontal lines represent the same chromosome for a panel of
lines. Identical stretches of marker scores (haplotypes) are indicated by the same colour. Box 1 indicates a region with depleted diversity whereas box 2 shows a highly
variable region.

assessment using chromosome haplotyping of lines, in (3) Breeding by Design

combination with phenotyping, provides the most power- Eventually, knowledge of the map positions of all loci of
ful route for optimally exploiting the germplasm of a agronomic interest, the allelic variation at those loci,
species. and their contribution to the phenotype should enable

A B C
1
2
3
4
5

2
x
3

F2
selection
Intermediate line I
x
1

BC
selection

Intermediate line II
x
5

BC
selection
Ideal line
R Sugars R Fruit mass I Plant Fruit color Cold tolerance Flowering Drought Fruit mass II
virus insect Fruit shape height time I resistance

TRENDS in Plant Science

Fig. 2. The principle of Breeding by Design. Subsequent selfings (F2) and BackCross (BC) selections using markers lead to the desired superior elite line genotype. Three
chromosomes, A, B and C, of five parental lines, 1 –5 are shown side by side. Specific recombination points are selected on chromosomes A and B whereas chromosome C
is selected from parental line 1. Dotted lines indicate marker positions used to select for the desired recombinants. Below the desired genome composition of the ideal line,
hypothetical resistance (R) and quality traits are mentioned.

http://plants.trends.com
334 Opinion TRENDS in Plant Science
the breeder to design superior genotypes comprising a
combination of favourable alleles at all loci. Because the
positions of all loci of importance are mapped precisely,
recombination events can be accurately selected using
flanking markers to collate the different favourable alleles
next to each other (Fig. 2). Software tools should enable us
to determine the optimal route for generating those mosaic
genotypes by crossing lines and using markers to select for
the specific recombinants that will eventually combine all
those alleles. Because this is a precisely defined process,
selection by phenotyping can be omitted. Only the even-
tually obtained superior varieties will be evaluated for
field performance.
Discussion
The above-described approach assumes several prerequi-
sites that require more discussion. (1) Extremely satu-
rated marker maps must be available to enable the
generation of high-resolution chromosome haplotypes.
Preferentially, a few markers are needed per window of
LD to ensure reliable high-resolution chromosome haplo-
types. Obviously, the extent of LD is strongly dependent on
several factors: among others, the crop species, the germ-
plasm selection and the genome region of interest [10].
Taking the rice genome as an example: assuming that
the average size of an LD window in rice is 100 kb and the
rice genome comprises 450 megabases, this means that
10 000– 20 000 mapped markers need to be scored on a
panel of inbred lines. This is feasible using the currently
available automation tools. Using a multiplex PCR marker
system such as AFLPw [13,14], generating 20– 100
markers per PCR, such a task can be performed within a
year with a few well-trained people and the appropriate
equipment. High resolution mapping of AFLP markers can
be performed using physical mapping strategies such as
Happy Mapping and Radiation Hybrid Mapping [15] or
applying BAC pooling strategies [16].
(2) The described approach requires extensive pheno-
typing of all agronomic traits, both the mapping popu-
lations and the inbred lines that are used for chromosome
haplotyping and allele assessment. Moreover, to map loci
involved in HETEROSIS , hybrids derived from selected
combinations of haplotyped lines must also be phenotyped.
The most important factor determining success is the
precision of the phenotyping. Dissecting the phenotypes
into components helps in objectively measuring pheno-
types: for example, a complex trait such as taste can be
dissected into sugar, acid and volatile flavour molecules,
which can be measured separately using biochemical
analysis equipment such as gas chomatographers. Extend-
ing this approach to all traits of agronomic importance will
require great organizational skills and will be a major
effort. In our view, the phenotyping is the most crucial and
challenging factor in achieving the goals set by Breeding
by Design.
Breeding by Design involves the integrative, comp-
lementary application of technological tools and the
materials currently available to develop superior varieties.
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Vol.8 No.7 July 2003
During this process, an enormous resource of knowledge is
generated that should enable breeders to deploy more
rational and refined breeding strategies in the near future.
The recent developments in technology and statistical
methodology have now brought this strategy within reach.
The optimal exploitation of the naturally available genetic
resources should create unsurpassed possibilities to gene-
rate new traits and crop performance. In our view,
Breeding by Design has as much crop improvement
potential as GMO strategies while requiring less invest-
ment and without challenging public acceptance.
Acknowledgements
We gratefully acknowledge Maarten Koornneef (Wageningen University
and Research Center), Jonathan Crouch (ICRISAT), Tom Gerats
(University of Nijmegen) and the comments of two anonymous reviewers
for their contributions and discussions during the development of this
paper. The AFLPw technology is covered by patents and/or patent
applications of Keygene N.V.; AFLPw is a registered trademark of
Keygene N.V.; A trademark registration for Breeding by Design has been
filed by Keygene N.V.
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