The Plant Journal (2002) 31(2), 211222

TECHNICAL ADVANCE

A novel method for the construction of genome wide

transcriptome maps
Bart Brugmans*, Asun Fernandez del Carmen, Christian W. B. Bachem, Hans van Os, Herman J. van Eck and
Richard G. F. Visser
Laboratory for Plant Breeding, Department of Plant Sciences, Wageningen University and Research Centre, PO Box 386,
6700 AJ Wageningen, the Netherlands

Received 7 December 2001; revised 21 March 2002; accepted 25 March 2002.

*
For correspondence (fax +31 317483457; e-mail Bart.Brugmans@pv.dpw.wag-ur.nl).

Summary
Expression proling by cDNA-AFLP is commonly used to display the transcriptome of a specic tissue,
treatment or developmental stage. In this paper, cDNA-AFLP has been used to study transcripts
expressed in segregating populations from Arabidopsis thaliana and potato (Solanum tuberosum). The
genetic differences between the offspring genotypes are thus visualized as polymorphisms in
the transcriptome. We show that polymorphic transcripts can be used as genetic markers and allow the
construction of a linkage map. The resulting map shows that, in contrast to genomic markers,
the transcriptome-derived markers did not cluster in particular areas of the chromosome, and that
cDNA-AFLP markers are targeted specically to transcriptionally active regions. The cDNA-AFLP markers
used in mapping are derived from DNA polymorphisms in transcripts, rather than differences in
expression regulation. The high potential of transcriptome markers as opposed to (anonymous) genomic
markers for applications in genetic analyses, marker-assisted breeding and map-based cloning is
discussed.

Keywords: cDNA-AFLP, DNA markers, transcriptome map, potato.

Introduction
Substantial efforts have recently been made to unravel the
DNA sequences of entire genomes (Wilson, 1999).
Complete sequence data are now available for numerous
species, including two small plant genomes, Oryza sativa
and Arabidopsis thaliana, with several other plant species
well on the way to completion (Arabidopsis Genome
Initiative, 2000). However, a wide range of other important
plant species, particularly those with large genomes, are
not likely to be comprehensively sequenced in the near
future. In such cases, one of the most productive
approaches to retrieving important genomic information
is the use of genetic mapping techniques to locate genes
responsible for a particular trait. Linkage maps from many
species have been established using various marker
systems. Besides RFLP and RAPD markers, the amplied
fragment-length polymorphism (AFLP) method is one of
the most widely used techniques. AFLP is a PCR-based
ngerprinting technique which efciently identies DNA
polymorphisms without prior sequence information (Vos
et al., 1995). AFLP relies on the selective amplication of a
subset of molecules from a more complex template pool.
There are many reports of AFLP technology being used for
the construction of genetic maps (Becker et al., 1995; van
Eck et al., 1995) or local marker saturation (Simons et al.,
1997). In most cases, maps are constructed for the
localization of genes responsible for qualitative or quan-
titative traits, as well as for positional cloning. For such
applications, however, the ideal distribution of markers is
not necessarily regular spacing across the whole genome,
but rather a concentration of markers in the coding regions
of the genome. As AFLP generally produces a distribution
of markers that is biased towards non-coding centromeric

2002 Blackwell Science Ltd 211

212 Bart Brugmans et al.
regions of the chromosome (Vuylsteke et al., 1999), this
may be regarded as a disadvantage for quantitative trait
loci (QTL) studies or positional cloning.
The cDNA-AFLP method is a robust method to generate
RNA ngerprints, and has been developed to visualize
gene expression (Bachem et al., 1996). By denition,
cDNA-AFLP targets only coding regions of the genome
and, like AFLP, it does not require prior sequence infor-
mation. Until now, cDNA-AFLP has been used for visua-
lization of differential gene expression and as a tool for the
isolation of genes (Bachem et al., 1998; Bachem et al., 2000;
Bachem et al., 2001; van der Biezen et al., 2000; Dellagi
et al., 2000; Durrant et al., 2000). Transcript-derived frag-
ments (TDF) obtained using cDNA-AFLP have also been
mapped by converting them into RFLP markers and linking
them to a genetic map (Suarez et al., 2000). Conversion of
cDNA-AFLP fragments into RFLP markers is, however,
time-consuming and prone to artefacts. Direct mapping of
transcripts with the cDNA-AFLP technique without further
conversion steps would overcome these problems and
result in a rapid and practical method of direct mapping of
expressed genes.
Information on expressed genes is currently being
collected in EST databases where the map position of
the gene is generally not known. Positional information on
genes is, however, essential for correlating mapped
phenotypes with genes at corresponding chromosomal
positions, allowing a candidate gene approach. An exam-
ple of a genome-wide candidate gene approach is pre-
sented by Chen et al. (2001), where sequences of known
genes were converted into PCR-based markers to map the
position of the candidate genes. For the success of this
approach, considerable knowledge is required of the
metabolic pathway and corresponding genes involved in
the development of the trait.
In this paper we describe a novel application of cDNA-
AFLP to visualize differences in the transcriptome between
offspring genotypes without prior sequence data. To our
knowledge, this is the rst application of a differential
display method for genetic analysis, as recently antici-
pated by Jansen and Nap (2001). Transcriptome variation
caused by the genetic differences between segregating
offspring genotypes allowed us to use individual tran-
scripts as genetic markers. To construct transcriptome
maps, we used diploid potato and A. thaliana. Diploid
potato is a non-inbred species where linkage analysis is
performed in progeny derived from crossing heterozygous
parents (Ritter et al., 1990), whereas in A. thaliana a
recombinant inbred line (RIL) population descending
from homozygous parents is available for mapping
(Lister and Dean, 1993) were used as examples of a simple
segregating population with homozygous parents.
Furthermore, we show that transcript markers do not
cluster in centromeric regions, and that most absence/
Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222
presence polymorphisms are based on single nucleotide
polymorphisms rather than on expression differences.
Results
Genetic diversity of the transcriptome visualized by
cDNA-AFLP ngerprints
The cDNA-AFLP ngerprints generated from potato shoot
tissue mRNA isolates contained between 35 and 112
amplication products with an average of 58 bands per
lane (36 primer combinations were tested). The fragment
sizes were distributed evenly between 50 and 500 bp.
Figure 1 shows a section of an autoradiogram image
where each lane represents a different genotype of a
potato mapping population including the parents.
Variation between the ngerprints was used to study
differences between genotypes at the transcriptome level.
The majority of the bands were monomorphic and did
not show marked variation in intensity between descend-
ants of a diploid potato mapping population. In contrast,
other TDFs showed large intensity differences that varied
from complete absence or levels just above the back-
ground to a signal strength that exceeded the capacity of
the phosphor screens. The observed intensity differences
are likely to represent >100-fold differences in gene
expression. As shown in Figure 1, the polymorphic tran-
scripts can be grouped into different classes. First, there
are absence/presence polymorphisms that show a 1 : 1 or
3 : 1 segregation ratio according to the mendelian models
ab 3 aa, aa 3 ab or ab 3 ab, where allele a represents the
absence of a band and allele b represents the amplication
of a TDF. These 1 : 1 and 3 : 1 segregating polymorphisms
can also be found in genomic AFLP ngerprints, and
suggest single-nucleotide polymorphisms (SNP) or inser-
tion/deletion (indel) events in the coding sequence of one
of the alleles. In addition to these absence/presence
polymorphisms, we also observed polymorphisms of
less pronounced intensity within the absence/presence
polymorphisms. The attributes of the polymorphisms
observed are shown in Table 1.
The total number of polymorphic transcripts that could
be scored per primer combination varied between one and
19, with an average of nine polymorphisms per lane. No
signicant correlation was observed between the GC
content of the selective nucleotides and the level of
polymorphism. In total, 331 polymorphic transcripts
could be unambiguously scored in the 90 offspring
genotypes and their parents. The reproducibility of the
cDNA-AFLP technique was veried by duplicating nger-
prints generated from seven genotypes sampled and
processed earlier for a pilot study (data not shown). The
reliability of the technique was also assessed by compar-
ing segregation data of cDNA-AFLP ngerprints with data
 Genetic analysis and linkage mapping using cDNA-AFLP 213

1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
500 bp

400 bp
A

300 bp 1 2 3 4 5 6 7 8 9 10
B

200 bp
1 2 3 4 5 6 7 8 9 10
C

1 2 3 4 5 6 7 8 9 10
D

100 bp

Figure 1. A section of a representative phosphor image.

Lane 1, size marker; lane 2, maternal parent; lane 3, male parent. The other lanes represent offspring from these two parents. A, Absence/presence
polymorphism originating from the male parent; B, intensity polymorphism; C, absence/presence markers heterozygous in both parents, a so-called
ab 3 ab marker; D, absence/presence polymorphism originating from the female parent with intensity differences between offspring with the band. On the
left, mobility is indicated as expressed nucleotides based on the size marker.

Table 1. Evaluation of genetic variation in the transcriptomes of potato siblings: description and number of cDNA-AFLP polymorphisms
in 36 TaqI/AseI primer combinations

Type of Absence/ Absence/ Absence/ Intensity

polymorphism presence presence presence variation Monomorphic
a
Genetic model ab 3 aa aa 3 ab ab 3 ab nd nd
Number 117 112 102 12 1733
Expected Mendelian ratio 1:1 1:1 1:3 1:0

a
Where allele a represents the absence of a band and allele b represents the presence of a transcript.nd, Not determined.

Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222

214 Bart Brugmans et al.

1 2 3 4 5

0 AseIAC/TaqITT-167
2 AseIAC/TaqITC-219 2 AseIAA/TaqITT-242
AseIAC/TaqITC-221
5 AseIAA/TaqITG-275 8 AseIAA/TaqITG-310 7 AseIAC/TaqITT-109
8 AseIAC/TaqITT-137 11 AseIAC/TaqITC-137
12 AseIAC/TaqITG-98 13 AseIAA/TaqITC-135

19 AseIAA/TaqITC-411
22 AseIAC/TaqITA-257
24 AseIAA/TaqITT-109

35 AseIAA/TaqITC-227
37 AseIAA/TaqITG-132
42 AseIAA/TaqITC-224

50 AseIAC/TaqITG-318
AseIAA/TaqITT-105 AseIAC/TaqITC-226
55 AseIAA/TaqITG-117 54 AseIAC/TaqITC-224
57 AseIAA/TaqITT-155 56 AseIAA/TaqITG-262
AseIAA/TaqITT-157
63 AseIAA/TaqITC-138

AseIAC/TaqITC-152
98 AseIAA/TaqITA-130
AseIAA/TaqITT-149

Figure 2. The Arabidopsis thaliana transcriptome map.

AseI/TaqI markers are cDNA-AFLP markers; RFLP markers from the map of Lister and Dean (1993) have been omitted to reduce the complexity of this
gure. The markers were used as a JOINMAP xed-order le for producing this map.

from anking loci obtained from genomic AFLP. No major
differences in location or distance between markers were
seen by adding cDNA-AFLP markers to the genetic map.
The potato mapping population used in this study
descended from a cross between highly heterozygous,
non-inbred diploid parents, which descend from vegeta-
tively maintained tetraploid cultivars that are known for a
high genetic load. Therefore a RIL mapping population
Col 3 ler of A. thaliana (Lister and Dean, 1993) was also
used to study genetic variation in the transcriptome of
homozygous siblings. RNA was isolated from Arabidopsis
seedlings 2 weeks after germination. Plant age was the
only criterion to obtain plant tissue of the same stage of
development. cDNA-AFLP ngerprints of Arabidopsis
resulted in one to seven clear absence/presence
polymorphisms per lane. These markers have been
added to the public marker data set of Col 3 ler (http://
www.arabidopsis.org.uk), and could be simply mapped.
Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222
This provides further evidence that transcriptome-based
markers obtained with cDNA-AFLP ngerprints of siblings
are as abundant and reliable as genomic markers.
Furthermore, the cDNA-AFLP markers from A. thaliana
were well distributed across all chromosomes of the map
(Figure 2).
Construction of a transcriptome map of potato
In order to establish a dense transcriptome map, TDFs with
clear segregation patterns were used to generate separate
maps of each parental set of gametes with JOINMAP 2.0
software. In total, 112 polymorphic transcripts descended
from the female parent; 117 from the male parent; and 102
transcripts were heterozygous in both parents ab 3 ab.
This resulted in 214 markers for the female and 219
markers for the male chromosome maps, which were
processed together with the data set of genomic AFLP and
 Genetic analysis and linkage mapping using cDNA-AFLP
RFLP markers of van Eck et al. (1995). At a logarithm of
odds (LOD) threshold of 4.0, 14 maternal linkage groups
were identied, and at a LOD threshold of 5.0 we identied
13 paternal linkage groups. The correct number of 12
linkage groups for each parent could be established by
connecting low LOD gaps within a linkage group via the
allele bridges with the homologous linkage group in the
other parent. The nal map agreed well with that obtained
previously (van Eck et al., 1995). The maps are shown in
Figure 3 and, in analogy with terminology such as
`isozyme map' or `genome map', we call this a `transcrip-
tome map'. The markers labelled AseI/TaqI are mRNA
polymorphisms, as visualized with cDNA-AFLP.
The map shows that the markers are scattered over all
the chromosomes. In four cases, a pair of markers of equal
mobility mapped to the same locus and differed only in
one selective nucleotide. Otherwise no obvious clustering
of cDNA-AFLP markers is found.
Origin of the transcript polymorphisms
The origin of absence/presence polymorphisms in tran-
scripts could be due to either genetic differences in gene
expression, or sequence polymorphisms in the coding
region. To determine which of these options was more
likely, internal primers were designed for four transcripts
that showed an absence/presence polymorphism, and RT/
PCR was performed on RNA from the genotypes showing
the cDNA-AFLP band polymorphism. All four primer pairs
were tested on undigested cDNAs of 10 offspring geno-
types, of which ve showed amplication of the TDF with
cDNA-AFLP. The resulting amplication products are
shown in Figure 4. All 10 offspring genotypes showed a
fragment of the expected length indicating that, irrespect-
ive of the cDNA-AFLP polymorphism, these genotypes
expressed the target gene.
The same primers were also used to amplify genomic
DNA of the mapping population. One of the resulting
amplication products had the expected length, and in
three cases intron-spanning primers gave a larger product.
Digestion with several restriction enzymes, including AseI
and TaqI, resulted in polymorphisms. The segregation of
the CAPS markers that were obtained from these four
cDNA-AFLP loci matched the segregation of the cDNA-
AFLP marker and therefore map at the same locus. From
this we conclude that the vast majority of the absence/
presence polymorphisms are due to genomic sequence
polymorphisms, probably SNPs at the sites of the selective
nucleotides or restriction sites essential for amplication
of the fragment with cDNA-AFLP. We conclude that the
map positions of transcript polymorphisms correspond to
the map positions of the genes from which they were
transcribed.
Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222
215
Sequence analysis
An arbitrary selection of 20 segregating cDNA-AFLP bands
has been isolated from gel, re-amplied and sequenced.
Subsequently, the sequences were analysed for homology
to genes in the sequence databases. For 18 transcripts,
signicant matches were found. Matches with an E value
of less than 1.0 3 105 were considered signicant. The
results are listed in Table 2. A review of the sequence
similarities shows that there is no functional or metabolic
relationship between the sequences similarities, suggest-
ing that the cDNA-AFLP transcript mapping method does
not produce a bias for a particular class of genes or for a
particular metabolic pathway.
Discussion
Here we describe a novel implementation of cDNA-AFLP
for the visualization of genetic variation within a mapping
population, and the targeted development of markers for
mapping the transcribed regions of the genome the
transcriptome. Jansen and Nap (2001) recently discussed
the potential uses of genetics combined with genomics.
They stated that the merger of expression proling and
genetic analysis will combine the power of two different
worlds in a way that is likely to become instrumental in the
further unravelling of metabolic, regulatory and develop-
mental pathways.
The experimental work described in this paper does not
completely support the opinion of Jansen and Nap. First,
our results show that the majority of the transcripts show a
monomorphic pattern, which indicates a high level of
uniformity of expression despite high levels of genetic and
phenotypic variation between these descendants.
However, phenotypic variation could also arise from
sequence polymorphisms that could not be visualized by
a single template. In contrast, hybridization-based gene-
expression analysis would completely fail to correlate
phenotypes with allelic versions of a gene that differ by
one or more SNPs in the coding region. Second, a
surprisingly low level (<1% of TDFs) showed clear quan-
titative variation in expression levels despite the wide
quantitative variation in the population. In order to detect
more differences in gene expression present in the popu-
lation, sampling of the RNAs would have to be more
restricted in terms of both cell type and developmental
range.
Consistency and reproducibility of the method
The good reproducibility of this method is demonstrated
by the fact that all cDNA-AFLP bands within the nger-
prints could be related to at least one parent, and that all
parental bands were inherited by offspring genotypes. The
216 Bart Brugmans et al.
many monomorphic bands demonstrate that reproduci-
bility across genotypes was good, and that the tissue
samples indeed represent the same developmental stage
(cDNA-AFLP ngerprints of other tissues can vary consid-
erably; data not shown). The reproducibility obtained in
this study is in agreement with results obtained from
cDNA-AFLP ngerprints from the potato tuber life cycle
(Bachem et al., 1996). Sequencing showed that cDNA-AFLP
bands represent bona de transcripts. Joint analysis with
data from genomic markers did not reveal inconsistencies
in transcript segregation patterns, or difculties in assign-
ment of markers to map position. The successful applica-
tion of this method on an A. thaliana mapping population
indicates that the construction of transcriptome maps
should be feasible for a wide range of organisms.
Genetic variation in transcript ngerprints
This paper describes genetic variation in the transcriptome
between descendants of a mapping population. The most
abundant and obvious polymorphisms between transcript
ngerprints of potato comprise the 1 : 1 and 3 : 1 segre-
gating absence/presence polymorphisms. These poly-
morphisms are predominantly based on SNPs or indels
in the transcribed sequence, and have also been observed
in genomic AFLP analysis (van Eck et al., 1995). Markers
arising from simple DNA polymorphism allow the corres-
ponding genes to be mapped directly. Evidence that this
polymorphism originates from cDNA sequence variation,
and not from expression variation, was obtained with
internal primers that could amplify the correct fragment in
all offspring, irrespective of the cDNA-AFLP polymorph-
ism. The co-localization of the cDNA-AFLP markers and the
derived CAPS markers provides additional conrmation
that this is a locus-specic marker system based on DNA
sequence polymorphisms of transcribed regions of the
genome.
Additional polymorphic patterns, unique to cDNA-AFLP,
are based on intensity differences that can, on occasion,
exhibit a difference of several orders of magnitude. These
intensity differences were also observed in combination
with absence/presence segregation. The clear-cut intensity
differences were rare, less than one per ngerprint, and
were not analysed here or used as markers for genetic map
construction. The origin of the observed variation in
intensity has not been investigated, but it can be
hypothesized that differences in promoter region or gen-
Figure 3. Maternal (C) and paternal (E) transcriptome maps of diploid potato.
The AseI/TaqI markers are cDNA-AFLP markers. Marker names ending with h are heterozygous in both parents, ab 3 ab, and allow connection to both
parental maps. Markers starting with TG are tomato RFLPs (Bonierbale et al., 1989; Tanksley et al., 1992). Markers starting with GP are potato RFLP markers
(Gebhardt et al., 1991). Markers starting with TPI, MDH, DIA, GOT and APS are isozyme loci (Jacobs et al., 1995). All the other letters and + stand for
different RFLP markers as described by Jacobs et al. (1995). The genomic AFLP markers from the map of Van Eck et al. (1995) are shown with an asterix
only to reduce the complexity of this gure.
Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222
etic differences in genes coding for trans-acting factors
cause the variation in expression level. Intensity poly-
morphisms could be mapped via QTL analysis, but the
locus obtained may correspond with either the gene
coding for the cDNA-AFLP band, or for the trans-acting
factor. A more detailed description of such cases has been
provided by Jansen and Nap (2001). The low number of
intensity polymorphisms found in our experiments is likely
to be due to the fact that whole plants were used for
sample preparation, obscuring the differences in gene
expression between cell and tissue types.
Map construction and marker distribution cDNA-AFLP
markers were easily mapped along with genomic markers
from previous studies (van Eck et al., 1995; Lister and Dean,
1993). The Arabidopsis cDNA-AFLP markers could be
placed with LOD values >3. For potato, separate maternal
and paternal maps were established. The 110 maternal
cDNA-AFLP markers could be placed with LOD values >4,
and 108 paternal cDNA-AFLP markers could be placed with
a LOD threshold >5. This indicates that cDNA-AFLP mark-
ers have power in genetic analysis equal to any other
molecular marker type currently used.
Most markers have a unique segregation pattern, which
resulted in a uniform distribution of cDNA-AFLP markers
along the chromosomes of potato. Inspection of the raw
data did not show ambiguities (singletons) for the place-
ment of cDNA-AFLP markers. In the maternal map (C) of
potato, three marker pairs were found (i) with the same
mobility; (ii) with only one difference in selective nucleo-
tides; and (iii) at the same map position. These marker
pairs could be alleles of the same locus, or represent
repeated sequences at the same locus. A further possibility
is that such markers may be generated by mispriming;
however, it has been shown previously (Bachem et al.,
1998) that mispriming events occur only very rarely in
connection with highly abundant transcripts.
Estimate of the number of transcribed genes
The number of fragments that are amplied with cDNA-
AFLP allows a rough estimate to be made of the total
number of genes of potato. In total, 2085 fragments were
visualized using 36 primer combinations, which is an
average of 58 per combination. All 256 + 2/+2 AseI/TaqI
primer combinations would visualize 14 848 fragments. A
recent analysis of restriction site frequencies using GenEst
(Qin et al., 2001) shows that in potato the AseI/TaqI enzyme
 Genetic analysis and linkage mapping using cDNA-AFLP 217

C1 C2
0 *
0 *E32/M48-360H

7 * 6 AseIAA/TaqITA-30 8 E2
8 AseIAT/TaqI AA-134 * 0
TG20B 2
12 * **
14 ***
AseICT/Ta qIAA-149H 7
15 **AseIAT/Ta qIAA-125H 16 AseIAA/TaqICA-309
16 ** *TG31 10
18 TDS168 TG9B
17 **
20 ** * 13
24 **** E1 23
24
AseIGG/Taq IAA-078
AseICG/TaqIAA-298
TG01B 14
25 **** * 0 **
27 *** 28 AseIAA/TaqICG-110 *E32/M48-360H 15
30 *AseITT/Ta qIAA-120H 32 **+ * 20
33 + * 22
34 AseIAA/TaqIGT- 340 + 8 35 ++
37 AseIAA/TaqICT-0 03 ****+ 23
* 11 36 * AGA/CAT-194.9H
40 AseIAA/TaqIGA-147 38 *
41 *SSP50 AseIAA/TaqITA-08 1
40 *** AseITG/TaqIAA-265H
43 TDS109 42 ** 27
45 TG24C1 AseIAA/TaqIAG-420
49 + SSP50 20 48 * AseICT/Ta qIAA-152H
51 * TDS109 21 50 + AseITT/TaqIAA-127 29
52 ** 51 ME(SOLANUM) AseITG/TaqIAA-266
53 S(SOLANUM) S(SOLANUM) 26 54 *TG01B AseIAA/TaqIGC-17 3H 31
AseIGT/Ta qIAA-083 56 *AGA/CAT-194. 9H AseIAC/TaqIAG-11 6 33
54 61 *
AseITA/TaqI AA-190H AseICA/TaqIAA-376
55 AseICG/TaqIAA-177 63 * AseIGT/TaqIAA-319H 34
58 ** AseIAA/TaqIGG-20 9 35 AseIAA/TaqIGC-234
61 * + 36
ST19 AseICT/Ta qIAA-289
62 * *AseIAA/TaqIG G-210H 41 65 AseIAA/TaqITC-2 34H
63 ** TPI-1 43 AseICA/TaqIAA-373H AseIAC/TaqIAG-23 7H 37
65 * * 44 AseITT/TaqIAA-152H AseICA/TaqIAA-373H
68 * ** 46 AseICA/TaqIAA-371 + 38
72 * * 47 69 * 42
AseICC/TaqIAA-423
TDS434
73
***AseIAA/TaqITA-28 7H 52 71 TG20B * 43
77 AseITG/Ta qIAA-112 ** 52
80 * + 53
73 AseIAC/TaqIAG-246
AseIAA/TaqICG-170 56 AseIAC/TaqIAG-23H * 55
85 AseIAT/TaqI AA-175 * 57 * 61
AseIAA/TaqITG- 142 76 +
88 78 * *ACA/CAC-266.0H
AseIAA/TaqICG-142 AseICG/TaqIAA-147 61 AseIGC/TaqIAA-20 2 67
90 AseITG/Ta qIAA-247 82 **
83 *ACA/CAC-266.0H AseICT/Ta qIAA-135H
93 AseICT/Taq IAA-324 * 68
94 * 85 AseITG/Ta qIAA-110
96 AseIAA/TaqICA-126 AseIAA/TaqIGC-173 H TDS250 70
97 AseIAA/TaqIGG-20 8 86 AseITG/Ta qIAA-121 AseICC/TaqIAA-565 72
100 AseICC/TaqIAA-258 87 + AseICT/Ta qIAA-269 73
103 AseITG/Ta qIAA-449 89 TDS441 AseICA/TaqIAA-219
AseIAC/TaqIAT-3 62 91 + *TDS441 74
105 + 75
AseIAA/TaqIGG-21 0H 92 AseICA/TaqIAA-154
108 TG53 94 * TDS434 79
109 + 99 *
113 TG259 106 *TG48

C4 E4
E3 0 *
*
*
0
3
AseIAA/TaqIGC-257 0 8 *AAA/ACG-196.0H SSP27 4
C3 TG130 1 14 AseIAC/TaqIAG-132 TDS436 14
* AseIAA/TaqITC-1 73 TDS374 17
0 AseIAT/TaqI AA-152 ** 8 17 AseICT/Taq IAA-150 20
AseIAA/TaqICC-172 AseICG/TaqIAA-251 H
**** 9 18 TG123 AseICG/TaqIAA-260 21
* 12 24 * AseICA/TaqIAA-107 23
6 * TDS258 13 28 * *AseIAC/TaqICT-27 4 24
9 *E32/M50-480H TI5-9 14 32 * AseIAA/TaqIAA-248 26
AseIAC/TaqIAC-106H 34 * AseICA/TaqIAA-414 27
15 *** E32M50-480H 16 36 + AseIGC/TaqIAA-229 32
AseIAA/TaqITT -099H 37 * AseICG/TaqIAA-205 H
16 *** * 35
*AseIAA/TaqIT C-300H 40 AseITG/Ta qIAA-245
18 *E39/M60-16H * 19 AAA/ACG-196.0H 38
**AAA/ACG-140.7H 41 AseITG/Ta qIAA-186 * 41
21 SSP66 AseIAA/TaqIAT-18 9
AseIAA/TaqIGT- 256H *AAA/ACG-140.7H 23 * 43
27 * 45 **AseICG/Taq IAA-251H + 44
AseIGA/TaqIAA-160 24 46 TDS374 + 46
29 TG08B AseIAA/TaqIAG-269 27 47 * * 48
30 *TG130 AseIAG/TaqIAA-111 29 48 * * 49
31 *E45/M60-26H AseIAA/TaqICA-151H 49 * ** 53
33 TI5-9 E45M59-153H 30 TDSG60 *AseIAC/TaqIAT-224 54
34 Y(SOLANUM) AseIAC/TaqIAG-348 50 TDS436 *
37 * *AseIGG/TaqIAA-265 32 * 58
AseIAC/TaqIAT-2 23 * 61
38 SSP66 E45M60-26H 33 53 *
+ 35 * 62
41 * 54 *E32/M48-165.4H * 65
44 TDS258 * 39 55 ** * 67
46 *TG134 **** 41 56 * **E32/M48-165.4H 68
47 TG42 * 44 59 * ** 69
50 + ** 46 61 * AC11-17 71
+ * 47 62 * AC8-99 72
51 * 48 65 AseIAA/TaqIAC-311 AC8-19
+ * 51 AC8-22 73
54 * 66 AseICG/TaqIAA-269 ** 75
** 55 68 AseIAA/TaqICG-220
58 + * 56 +ADH-2 77
71 AseIAC/TaqIAC-125 AseIGC/TaqIAA-327 H
E45/M59-153H * 57 73 AC11-17
AseIAT/TaqI AA-434 ** 78
62 AseIAC/TaqICT- 309 59 Ac8-99 *
*AseIAA/TaqI CA-151H AseIAA/TaqIAG-260 60 74 Ac8-19 81
*
65 AseIAA/TaqIAA-175 AseIAA/TaqICA-410 61 75 Ac8-22 * 83
AseIGT/TaqIAA-205 63 77 **** + 87
70 + AseIAC/TaqIAG-341 65 78 * * 88
73 + **E39M60-16H 67 79 *AseIGC/TaqIAA-327H + 90
AseIGA/TaqIAA-286 70 AseIAC/TaqIAC-077 91
75 AseIAC/TaqIAG-29 6 81 * AseIAA/TaqIAA-379 92
AseIAC/TaqIAC-349 72 85 AseITC/Taq IAA-118 AseIAG/TaqIAA-138 93
** 75 89 AC11-13 AseITT/TaqIAC-182 94
* 82 90 TI31 AC11-13 95
93 *+ TI31 96
96 BE TG123 99
97 TG22 TG22 102
98 TI17 BE 103
TDS69 TI17 105
101 AseICG/TaqIAA-122 TDS168 106
103 AseIAA/TaqIAA-376

Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222

218 Bart Brugmans et al.

E6
E5 C6 **** 0
TDS259 0 0 GP79
*** 1
*** 2
C5 5 AseIAC/TaqICT- 263 * 6
0 * * 8 9 AseIAC/TaqICT- 400 *** 7
5 GP21 AseIAC/TaqIAG-41 0 ** 10
EL(SOLANUM) 9 AseIAA/TaqITC- 138H
6 AseIAA/TaqITG -490 * 10 12 ** 12
AseIGG/Taq IAA-138 * 13
7 AseIAA/TaqITG -492 GP21 15 AseIAC/TaqIAC-179 H
8 + AseIAA/TaqITA-2 42 18 16 TG231 TG118 16
9 AseIAA/TaqIAC-275 AseIAC/TaqICT -260 19 17 ***+ AseIGT/TaqIAA-101 18
15 AseIAC/TaqIAC-27 7 AseIAT/Taq IAA-460 21 18 *
17 * + 23 21 TG118
** 24 * 25
19 *AC4-68 + 27
* 25 27 ADH-1
20 **E32/M61-15H TG44B 28
21 **E32/M48-252.4H * 28
+ 31 ** 30
22 * 35 +
** 32
24 ** * 33 37 AseIAC/TaqIAA-285
25 ** AseIAA/TaqICG-24 1H TG253 36
**E32/M48-252.4H 34 38 AseIAA/TaqITG- 180
27 *E32/M51-21H + 37 * 39
28 * * 38
29 * **E32/M51-21H 41 46 TG253 ** 46
30 ** * 43 + 47
35 *** 49 +
* 44 * 49
37 TDS416 *E32/M61-15H 45 53 TDS183 + 50
39 + SSP88 46 AseIAA/TaqIGG- 232 51
*TDS416 48 56 +
40 *SSP88 AseIAA/TaqIAT-3 11 53
45 * + 51
* 53 61 TG193 AseICA/TaqIAA-122H 54
47 GBSSB AseICA/TaqIAA-140
49 * * 56 *
AseIAA/TaqITA-2 40H 60 65
AseICA/TaqIAA-122H
AseIAA/TaqIAA-212 57
50 *AseIAA/TaqIT T-2 72 AseIAT/Taq IAA-207 61
52 MDH-2 *AseIGA/TaqIAA-330 62
AseIAA/TaqICA-338 64 TG193 63
53 *DIA-1 AseIAA/TaqIAA-510 68 * 66
AseIAA/TaqIAA-367 TG23 69 TDS183 68
55 CHSST GBSSB 70
57 ** * 74
59 TG69 YM(SOLANUM) 76 C8
60 * AseIAA/TaqIAA-315 78 0 GBSS
61 * CHSST 82 2 AC15-8 E8
4 + GBSS 0

C7 10 +
0 * 14 TDS458 AC15-8 7
16 TDS385
AseICA/TaqIAA-144 TDS458 12
25 ASEIAA/TAQIAG-317H TDS385 14
8 * AseIAA/TaqITA-1 63H
13 + AseIAA/TaqIGG- 181 * 17
16 * E7 ** 19
18 * 30 AseIAA/TaqITG -094H
AseICG/Taq IAA-241 **** 22
21 * AC4-62 0
22 * + AseICA/TaqIAA-089 25
24 AseIAA/TaqICA-152 * 4 31 * AseIAC/TaqIAG-1 14 26
AseIAT/Taq IAA-411 AseIAC/TaqIAC-12 1 **** 32
27 * 7 * 33
AseITT/TaqI AA-411 32 AseIAA/TaqIGT -359
AseIGC/TaqI AA-410 + 10 35 AseICA/TaqIAA-164 GOT- 1 35
29 * 36
AseIGT/TaqIAA-410 37 *TG16A
32
*AseIAA/Taq ITA-38 0H TI107 16 40 * *AseIAA/Taq IAA-105 38
TG143 AseIAA/TaqIAT-3 24 20 41 * * 39
34 ** AseICA/TaqIAA-087 *** 40
35 + 21 43 TDS429
AseIAA/TaqIGG-5 60H 45 ** *E32M48-451.7H 41
36 ** AseITG/TaqIAA-143 22 * 42
38 NBS17 46 * * 43
AseICA/TaqIAA-090 47 **
39 * AseIGG/Ta qIAA-324 23 * 46
41 NBS9 AseIAA/TaqITG- 560H 50 TG45 * 47
47 TG572 52 *** TDS429 48
53 AC4-62 AseIAA/TaqIGC-08 8 24
TG438H 53 * TG45 53
54 * 54 *
56 TI107 AseIAA/TaqICT- 004 30
+ 32 56 AC3-28 AC3-18 60
58 *
59 * AseIAC/TaqIAA-217 33 57 *
60 **** + 35 58 ***E32/M48-451.7H
AseIAA/TaqITA-3 75 36 63 * APS-2 66
62 *
63 ** TG61 39 66 AseIGA/TaqIAA-185
67 GOT- 2 * 45 68 AseIAA/TaqITG -510 CO(SOLANUM) 71
69 ** TG63B 48 70 AseIAC/TaqIAC-08 9
75 * 72 *
76 AseIAA/TaqIGG-2 66 75 *
78 AseIGA/TaqIAA-151 78 *
79
AseIAC/TaqIAA-122
AseIGC/TaqI AA-351
82 AseIAA/TaqITA-3 69
87 AseIAA/TaqIAT-3 33
AseIAA/TaqIAT-3 40H
88 **
89 *
90 *

98 *
102 *

Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222

 Genetic analysis and linkage mapping using cDNA-AFLP 219

E9 E10
AC38-46 0
*TG09 0 TDS448 6
* 8
* 4 * 12
* 6 * 14
TAC93A 8 ++ 17
* 10 + 21
C10 * 23
* 13 0 TDS448 TG43 26
**** 29
+ 18 ** 31
+ 20 6 ST15B *E32/M47-397H 33
C9 AseIAG/TaqIAA-680 H 23 * 35
0 AseIAA/TaqIAG-077 10 ST06 **E32/M54-153H 36
1 TG10 AseIAT/Taq IAA-223 25 ** 39
AseIAT/Taq IAA-222 28 12 + *TDS273 40
AseIAA/TaqIAA-415 33 * 41
AseIAG/TaqIAA-408 16 + *** 43
8 *TG09 AseIAA/TaqITG- 118 35 18 *AC15-7 ** 45
AseIAA/TaqIGG-1 18 36 21 TG63 * 49
***E45/M60-7H 50
*AseIAC/Taq ICT-2 30 38 ** 54
15 ** AseIAC/TaqIAA-200 41
AseIAA/TaqIAT-18 6H * 55
16 TAC93A AseIAA/TaqIAA-345H ****AseIAC/TaqIAG-168H 56
AseIAC/TaqIAA-308 43 31 AseIGT/TaqIAA-220H + 57
21 TG35 AseIAA/TaqITA-1 98H AseIGT/TaqIAA-221 **TG303 58
23 * 32 AseIAA/TaqIGC-13 7H AseIAA/TaqIAA-149 60
AseIAA/TaqIGT- 177H 45 AseIAA/TaqITT -128 AseIAA/TaqITC-3 15 64
24 *****E32/M51-26H AseICT/Ta qIAA-156
36 E32/M54-153H AseIAC/TaqIAC-369 65
AseICG/TaqI AA-326 *E32/M51-7H AseIAC/TaqIAT-1 49H
AseIAA/TaqIAT-3 20 49 67
30 * 40 + AseIAG/TaqIAA-246
AseIAA/TaqITA-1 90 53 AseIGT/TaqIAA-340 69
* 57 AseIAT/TaqI AA-335
35 **AseIGT/TaqIAA-165 *TG35 60 AseIAA/TaqIAT-13 3 72
50 *** AseICG/TaqIAA-460 74
39 **TG06 * 63 ** 76
42 * *** 66 51 TDS273 * 77
43 * * 67 * 79
44 * + 69 57 * AseIAA/TaqITG- 240
**** 70 58 * AseIAA/TaqITG- 246 82
46 * AseIAA/TaqITA-41 6 83
47 * *+ 72 AseICC/TaqIAA-136 87
* 74 65 + * 88
55 AseIAC/TaqIAC-213 * 76 AseIAA/TaqIGC-137 H
58 AseIAA/TaqIAT-1 05 66 +
*TDS369 77 AseIAA/TaqIAT-18 6H
61 *TDS396 68 AseIAA/TaqIGT- 115 AseIGT/TaqIAA-220H 89
* 78
62 AseIAC/TaqIAA-137 **AseIAC/TaqIAA-20 5H 79 AseIAA/TaqIGG-2 14
AseIAC/TaqIAA-138 AseIGT/TaqIAA-219 94
* 80 75 *E32/M47-397H AseIAG/TaqIAA-242H
63 AseIAC/TaqIAA-205H * 83 77 TG303 AseIAG/TaqIAA-245 95
AseICA/TaqIAA-131H ** 84 AseIAA/TaqIGG-2 41 99
66 * *** 85
67 * *E32/M51-26H 86 E12
68 + * 91 C12 AseIAA/TaqICG-37 9 ST14 0
69 AseIAA/TaqITG -160 ** 94 AseIAA/TaqIAC-328H ST20 4
** 95 AseIAA/TaqIGC-38 9H **TDS319 10
* 96 0 AseIAC/TaqIAA-422H TDS103 12
* 97 AseICG/TaqI AA-098H AseIAA/TaqIGG-1 54 13
* 102 AseITG/TaqIAA-259H AseIGA/TaqIAA-431 15
+ 103 AseIAA/TaqIAA-166 16
1 AseIGC/TaqI AA-274 * 17
* 104
* 111 8 TG360 E32/M54-182H
9 * E32/M59-109H
C11 10 AseITG/TaqIAA-281 ***E32/M59-162H 19
13 ST14 **E32/M48-416H
0 AseIAA/TaqITA-0 73 ACA/CAC-276.5H
1 AseITG/TaqIAA-277 E11 18 ST20 AAA/ACG-278.0H
* 0 21 TDS319 ** 20
5 AseICA/TaqIAA-086 23 TDS103 **** 21
26 * **** 22
28 TG68 ** 24
11 * *ACA/CAC-276.5H *E45M60-5H
13 * 30 E32M51-33H 27
E32M59-109H
33 + *+ 28
17 * + 29
19 * ** 14 35 **
*E32M54-182H * 33
AseIAA/TaqIGA-209 * 15 ** 34
21 * 16 36 E32M48-416H
*AseIAA/Taq ICT-0 02H + 35
E45M60-5H
26 AseIGG/Ta qIAA-203 ** 18 37 *
* 37
* 22 ** 39
30 AseIAC/TaqIAA-166 39 ****E32M59-162H * 42
31 * *AseIAC/TaqI AC-400 25 E32M51-33H **** 43
32 * P(Solanum) 40 *AAA/ACG-278.0H * 44
+P(SOLANUM) 29 42 * + 48
33 AseICT/Ta qIAA-157
36 TG44 AseIAA/TaqIAT-2 43H 48 AseICG/TaqI AA-301 **** 52
38 *** AseIAA/TaqIAC-178 30 49 * ** 54
51 AseITT/TaqI AA-084 AseIAG/TaqIAA-224 59
42 *+ AseIAG/TaqIAA-216 32 52 **AseIAT/T aqIAA-170H AseIAA/TaqIGC-18 0 61
43 *** AseIAC/TaqIAG-3 38 36 +E32M48-154.2H AseIAA/TaqIGT- 210 62
TG44 38 55 AseICG/TaqI AA-226 AseIAT/Taq IAA-092 63
47 TG30 * 64
AseIAA/TaqIGT- 096H * 66
52 SSP75 60 AseIAC/TaqIAG-15 0 + 69
55 AseIAA/TaqICG-35 0 SSP75 44 62 ****ACA/CAC-247.1H PGDH-2 70
56 AseIAA/TaqICC-168 64 + AseIAT/Taq IAA-380
65 TG491 AseIGA/TaqIAA-150 H 71
57 AseIAA/TaqITC- 158 TG30 49 66 + ** 72
71 TDS163 AseICT/Ta qIAA-236
63 AseIGG/Ta qIAA-120 78 AseICC/TaqIAA-165 AseIAA/TaqIGC-21 9H 74
64 AseIGC/Taq IAA-161 AseIAA/TaqIGT- 096H
66 AseIAT/Taq IAA-271 AseIAA/TaqIAC-378
AseIAT/Taq IAA-170H 78
* 79
**E32/M48-154.2H 81
*ACA/CAC-247.1H 82
TG491 85
* 90
TDS163 91

Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222

220 Bart Brugmans et al.

combination is likely to cut only around 24% of all full-
length cDNAs and that, on average, these cDNAs produce
1.7 cDNA-AFLP fragments. This gives rise to an estimated
number of transcribed genes in the aerial plant tissues of
1 2 3 4 5 6 7 8
around 36 000, which is similar to previous estimates
(Bachem et al., 2000).
This estimate in potato exceeds the estimate of the
number of genes of A. thaliana (Arabidopsis Genome
Initiative, 2000). Ku et al. (2000) made a calculation about
the total number of genes in tomato, another Solanaceae
species. These calculations are based on the similarity
9 10
between A. thaliana and the sequence a 105 kb bacterial
articial chromosome of tomato. They suggested that the
number of genes could be up to 145 000 genes. Our results
only suggest that there may be more than the 25 000
genes of A. thaliana.

Application of cDNA-AFLP transcript polymorphisms

Figure 4. RTPCR of transcripts corresponding to four segregating TDFs.
The origin of four 1 : 1 segregating absence/presence cDNA-AFLP
markers is caused by sequence polymorphism affecting the cDNA-AFLP
amplication product. PCR amplication of RNA from 10 offspring, using
internal primers, demonstrates the presence of these four transcripts in
the steady-state RNA pool.
In this study we have focused on the application of
transcript polymorphisms for marker development and
the construction of a transcriptome map. We have shown
that cDNA-AFLP markers could be converted in CAPS
markers. CAPS markers are more easily applied in marker-
assisted breeding, but CAPS markers derived from cDNA-
AFLP have the advantage that they specically target
transcribed genes.
In future studies we plan to analyse transcripts with
quantitative variation in expression level in more detail.
For example, we wish to understand whether this expres-

Table 2. Accession numbers, E values and annotated function of sequences with the best homology to potato cDNA-AFLP fragment
sequences excised from transcript ngerprints
a
BLASTx TBLASTx

cDNA-AFLP Accession Accession

fragment number E value number E value Annotated function

AseIAA/TaqIGC-235h AI899537 4.00E-29 Coatamer required for vesicle budding and anterograde
vesicle transport from and to the Golgi
AseIAA/TaqIGC-139c Unknownb
AseIAA/TaqIGC-137h AI775597 3.00E-09 Unknown
AseIAA/TaqIGC-135h AI775597 2.00E-05 Unknown
AseIAA/TaqIGG-540h P36181 8.00E-19 HSP80 homologue (heat-shock protein)
AseIAA/TaqIGG-214e AJ133755 1.00E-09 Peptidyl-prolyl cis-trans isomerase (required for mitotic cell
cycle progression)
AseIAA/TaqIGG-169h AJ132397 7.00E-06 Major latex protein
AseIAA/TaqITC-445h AJ222713 4.00E-18 NAP gene homologue (no apical meristem)
AseIAA/TaqITC-344c Unknownb
AseIAA/TaqITC-315e Unknownb
AseIAA/TaqITC-173c T05027 1.00E-05 Reverse transcriptase
AseIAA/TaqITC-158c Unknownb
AseIAC/TaqIAA-418h M59857 6.00E-60 Stearoyl-acyl carrier protein desaturase
AseIAC/TaqIAA-358h P51414 4.00E-14 Ribosomal protein
AseIAC/TaqIAA-217e Unknownb
AseIAC/TaqIAA-205h AJ237988 4.00E-07 Putative ripening-related protein
AseIAC/TaqIAA-200h BF459608 2.00E-18 PG type gene (fruit ripening)
AseIAC/TaqIAA-138c Unknownb

a
TBLASTx was carried out using the `EST others' database, only in cases where the BLASTx yielded no signicant homologies.
b
cDNA-AFLP fragments yielding no homologies with any searches.

Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222

 Genetic analysis and linkage mapping using cDNA-AFLP
sion variation can be used to map the locus of transcrip-
tion factors. Furthermore, we intend to correlate pheno-
typic variation with these quantitatively and qualitatively
segregating transcript markers in a genome-wide exten-
sion of the candidate gene approach. Finally, we wish to
exploit this technique for map-based cloning. BAC landing
is currently achieved with genomic markers, but local
saturation of a map with bulked segregant analysis of
cDNA-AFLP markers would increase the efciency of BAC
landing, possibly into `gene landing'.
Experimental procedures
Plant material
A diploid mapping population of 90 F1 diploid potato genotypes
descending from the non-inbred parents USW5337.3 (clone
C) 3 77.2102.37 (clone E). Clone E was obtained from a cross
between clone C and VH34211, and therefore this F1 population
resembles a back-cross. This mapping population has been used
in previous genetic studies (Jacobs et al., 1995; Van Eck et al.,
1995), and has been maintained in vitro for several years. For this
experiment, shoot cuttings were put on MS medium (Murashige
and Skoog, 1962) and harvested after 4 weeks, with all plants as
much as possible in the same stage of development. Shoot tissue
(15 g) was harvested and ground to a ne powder under liquid
nitrogen and stored at 80C until RNA isolation. The Arabidopsis
RILs and their parents were sown on MS20 medium (Murashige
and Skoog, 1962) and seedling tissue was harvested after
2 weeks. No specic efforts were taken to make sure that the
lines were in the same state of development. The plants were
harvested and ground under liquid nitrogen and stored at 80C
until RNA isolation.
mRNA isolation, cDNA synthesis and template
preparation
Total RNA was isolated from the ground plant material. The total
RNA concentration was estimated by visualization on a 1%
agarose gel. The poly(A)+ RNA fraction was extracted from
10 mg total RNA using poly(d)[T] 25 V oligonucleotides coupled to
paramagnetic beads (Dynal A.S., Oslo, Norway). First- and
second-strand cDNA synthesis was carried out according to
standard protocols (Sambrook et al., 1989). The volumes for
mRNA isolation and cDNA synthesis were adjusted to facilitate
handling in microtitre plate format such that 92 samples could be
processed simultaneously. The nal volume after cDNA synthesis
was 50 ml. Then 5 ml of the reaction mix was analysed on a 1%
agarose gel to estimate the concentration, which was equalized to
10 ng ml1. Of this, 25 ml was subjected to the standard AFLP
template production (Bachem et al., 1996). Restriction enzymes
used for template preparation were AseI and TaqI. Further steps
to provide template for cDNA-AFLP were carried out as described
previously (Bachem et al., 1996). All amplication reactions were
done on a PE-9600 thermocycler using Taq DNA polymerase (PE
Biosystems, Foster City, CA, USA). All oligonucleotides used in
the cDNA-AFLP procedure were obtained from Eurogentec
(Seraing, Belgium). All enzymes were from Life Technologies
(Gaithersburg, MD, USA) with the exception of AseI and TaqI,
which were from NE-Biolabs Inc. (New Brunswick, NE, USA).
Blackwell Science Ltd, The Plant Journal, (2002), 31, 211222
221
Radioactive cDNA-AFLP and gel analysis
Radioactive labelling of primers was carried out using g33P-dATP,
and conditions for PCR were according to Bachem et al. (1998).
Samples were denatured and separated on 4.5% polyacrylamide
sequencing-type gels. Gels were transferred onto Whatman 3MM
paper and dried using a slab gel dryer. After 20 h exposure to a
phosphor screen, the labelled DNA fragments were visualized by
phosphor imaging using STORM 860 (Amersham Pharmacia
Biotech, Uppsala, Sweden). The gels were also exposed to X-ray
lms and developed after 3 days' exposure.
Data analysis and map construction
Data on transcript polymorphisms were collected by visual and
quantitative interpretation of autoradiograms and outputs from
the phosphor-imager (STORM 860, Amersham) by the image-
interpretation software CROSSCHECKER (Buntjer, 2000; http://
www.dpw.wau.nl/pv/pub/CrossCheck/download.html). In total,
36 random primer combinations were used to ngerprint the
potato population of 90 individuals and two parents. For
Arabidopsis, only eight random primer combinations were used
to ngerprint the RILs. Marker nomenclature is based on the
restriction enzymes used to generate cDNA-AFLP template, the
selective nucleotides of the primers and the mobility of the band
relative to a 10-base ladder (SequaMark, Research Genetics, San
Diego, CA, USA). The additional letter c, e or h included in the
potato marker name indicates whether the marker locus is
heterozygous in the maternal or paternal parent, or both.
Segregating transcripts were assigned to map positions with
JOINMAP 2.0 (Stam, 1993), along with genomic marker data of the
potato mapping population (Van Eck et al., 1995) or the
Arabidopsis mapping data (Lister and Dean, 1993). Linkage
group nomenclature is in agreement with existing Arabidopsis
(Lister and Dean, 1993), potato and tomato maps (Bonierbale
et al., 1989; Gebhardt et al., 1991). The maps were drawn with the
graphical package MAPCHART (Voorrips, 2000).
Isolation and analysis of transcript fragments
Twenty randomly chosen transcripts comprising absence/pres-
ence polymorphisms, intensity polymorphisms and mono-
morphic transcripts were sequenced. Fragments were excised
from dried gel, put in 200 ml Tris-EDTA pH = 8.0 (TE) and left for
1 h. From this solution, 5 ml was taken for a 35-cycle PCR to re-
amplify the fragment. The resulting DNA was diluted to 30 ng ml1
and the nucleotide sequence determined (Eurogentec). The
sequences were analysed remotely (NCBI, Bethesda, MD, USA)
for homology to databanks using the BLAST 2.0 programs
(Altschul et al., 1997). The GenBank database `nr' was used for
BLASTx analysis and the database `EST others' for TBLASTx.
To assess the cause of absence/presence polymorphisms,
internal primers were designed on the following transcript
fragments: AseIAA/TaqIGG-214e; AseIAA/TaqITC-173c; AseIAA/
TaqITC-158c and AseIAC/TaqIAA-217e. With these primers a 35-
cycle PCR was performed on cDNA templates of ve samples that
did have the transcript and ve lacking the band on the cDNA-
AFLP ngerprint. The result of this PCR was separated on a 3%
agarose gel to determine if a fragment of the correct length was
present or absent.
To conrm that the map position of cDNA-AFLP markers
matched the map position of a DNA sequence polymorphism,
rather than the locus of a transcription factor, cDNA-AFLP markers
222 Bart Brugmans et al.
were converted to CAPS markers. The same primers were used to
amplify genomic DNA from descendants of this C 3 E population.
CAPS polymorphisms were obtained after digestion of the PCR
product with a series of four cutter restriction enzymes including
AseI and TaqI
Acknowledgements
We would like to thank the company Genetwister (Wageningen,
the Netherlands) for the use of their phosphor imaging facility.
We would also like to thank Yan Zifu for his assistance during
RNA preparations. Maarten Koornneef is acknowledged for
providing the Arabidopsis thaliana seeds of the Col 3 ler map-
ping population.
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