Molecular Breeding 13: 211–227, 2004.

211
© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Characterisation and genetic mapping of resistance and defence gene

analogs in cocoa (Theobroma cacao L.)

Claire Lanaud1,*, Ange Marie Risterucci1, Isabelle Pieretti1, Jeanne A.K. N’Goran2 and
Dominique Fargeas1
1CIRAD/BIOTROP, 2477 avenue Agropolis, TA40/03, 34398 Montpellier cedex 05, France; 2CNRA, 01 BP
1827, Abidjan 01, Côte d’Ivoire; *Author for correspondence (e-mail : claire.lanaud@cirad.fr; fax : (33) 4 67
61 56 05)

Received 28 January 2003; accepted in revised form 25 August 2003

Key words: Candidate gene approach, DGA 共defence gene analog兲, Disease resistance, Phytophthora, RGA 共re-
sistance gene analog兲, Theobroma cacao

Abstract

Disease resistance and defence gene analog 共RGA/DGA兲 sequences were isolated in cocoa using a PCR ap-
proach with degenerate primers designed from conserved domains of plant resistance and defence genes: the
NBS 共nucleotide binding site兲 motif present in a number of resistance genes such as the tobacco N, sub-domains
of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene fami-
lies: pathogenesis-related proteins 共PR兲 of classes 2 and 5. Nucleotide identity between thirty six sequences iso-
lated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated
from corresponding coding sequences produced sequences without stop codons, except for one NBS ⫺ like se-
quence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed
: NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in
five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different se-
quences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrich-
ment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs,
DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where
a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious
diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease
management.

Introduction species, P. megakarya is the most destructive, and is

Several Phytophthora species cause Black pod
disease in cocoa. One of them, P. palmivora 共Butler
1919兲 has a worldwide distribution and is known to
cause yield losses as high as 30%. The other species
are P. megakarya 共Brasier and Griffin 1979兲 in West
and Central Africa, and P. capsici 共Tsao and Alizadeh
1988兲 and P. citrophthora 共Babacauh 1980兲 in Latin
American countries. Among the four Phytophthora
frequently responsible for yield losses which can ex-
ceed 50%.
Quantitative resistance to Phytophthora has been
observed in Theobroma cacao. Based on phenotypic
observations, several authors 共Blaha and Lotodé
1976; Enriquez and Salazar 1980; Rodriguez et al.
1985; Tan and Tan 1990; Warren 1994; Enriquez and
Soria 1996; Cilas et al. 1996兲 have suggested that re-
sistance is under polygenic control.
212
QTL analyses have been carried out to study the
genetic basis of Phytophthora resistance 共Lanaud et
al. 1999; Risterucci et al. 2003; Crouzillat et al. 2000;
Lanaud et al. 2000; Flament et al. 2001兲 and have
confirmed the polygenic nature of cocoa resistance to
several species of Phytophthora. QTL associated with
resistance to single or several Phytophthora species
have been identified in the cocoa genome.
The individual genes contributing to the QTL for
quantitative resistance in plants are of limited
biological significance. Map-based cloning of such
genes is particularly labor-intensive and expensive.
An alternative approach is to focus on genes that are
potentially involved in the biochemical pathways of
disease resistance. This approach, called the candidate
gene approach, is based on a forward hypothesis that
certain genes of known function related to plant re-
sistance play a role in the plant–pathogen interaction
of interest. These genes are isolated in the studied
species and their locations compared to those of the
QTL. Their level of expression under disease pressure
is assessed, and finally their involvement in resistance
mechanisms could be validated by transformation ex-
periments.
Many genes involved in plant–pathogen interac-
tions have already been cloned. Genes conferring re-
sistance to plants belong to two main groups: one is
composed of genes involved in pathogen recognition
and/or signal transduction, and called R genes 共Ham-
mond-Kosack and Jones 1997兲, the other corresponds
to genes implicated in defence mechanisms, and their
product synthesis occurrs de novo in response to
pathogen recognition 共Bol et al. 1990; Stintzi et al.
1993兲. R genes belonging to the major classes of plant
pathogens have been cloned in various species. Se-
quence comparisons between these genes have
revealed structural similarities in functional domains,
making it possible to classify R-genes into four main
classes 共Hammond-Kosack and Jones 1997兲. One
class of genes encodes cytoplasmic receptor-like pro-
teins that contain a leucin-rich repeat domain 共LRR兲
involved in protein-protein interactions, and a nucle-
otide-binding site 共NBS兲. This class of genes includes
the tobacco N gene 共Whitham et al. 1994兲, the flax
L6 gene 共Lawrence et al. 1995兲 and the Arabidopsis
RPS2 gene 共Bent et al. 1994兲. Another class of genes,
the LRR group, which includes the tomato Cf genes,
possesses only an extracellular LRR region. A third
class of R-genes, the kinase group, contains a serine-
threonine kinase domain 共STK兲, involved in intracel-
lular transduction 共Bent 1996兲, either alone, as in the
tomato Pto gene 共Martin et al. 1994兲, or associated
with a receptor-like kinase structure, as in the rice
Xa21 gene 共Song et al. 1995兲 or in the wheat LRK10
gene 共Feuillet et al. 1997兲.
The other group of genes, the defence response
genes, plays a role in limiting pathogen invasion of
plant tissues, by limiting pathogen growth, develop-
ment, or propagation within the plant. PR 共Pathogen-
esis Related兲 proteins were first isolated from tobacco
共Gianinazzi et al. 1970; Van Loon et al. 1970兲, then
from various mono- and dicotyledons. Their synthe-
sis is induced by pathogens during the infection pro-
cess. They are classified according to their electro-
phoretic mobility and serological properties.
Biological properties have been defined for some cat-
egories of PR proteins, such as: glucanase activities
共hydrolysis of the polymers in fungal walls, elicita-
tion of plant cells and triggering defence mecha-
nisms兲 共Kauffmann et al. 1987; Farmer and Ryan
1992兲, chitinase, peroxidase, etc. The anti-fungal ac-
tivity of PR-5s has been shown in vitro on spore ger-
mination and mycelium growth in numerous patho-
genic species, including Phytophthora 共Woloshuk et
al. 1991兲. It has also been demonstrated in vivo by
Fagoaga et al. 共2001兲 after transformation of an or-
ange variety 共Citrus sinensis L.兲 susceptible to Phy-
tophthora citrophthora with a chimaeric construct
containing the coding region of the tomato pathogen-
esis-related PR-5. Moreover, Faris et al. 共1999兲 sug-
gested that many minor resistance QTL, such as those
identified in quantitative resistance, may correspond
to defence response genes.
Several strategies to search for candidate genes
could be developped. One of these could consist in
isolating genes that are expressed differentially in re-
sistant and suceptible plants during the infection pro-
cess. If EST databases are available for the species
studied, these could be exploited in the search for
orthologous resistance and defence genes. Alterna-
tively, a PCR 共polymerase chain reaction兲 strategy
could be used: the conserved domains of resistance
or defence genes offer the possibility of amplifying
DNA fragments analogous to resistance genes 共RGA兲
or to defence genes 共DGA兲 in a new species using
degenerate primers 共Leister et al. 1996; Kanazin et al.
1996兲 or combining the use of degenerate primers
with AFLP techniques 共Hayes et al. 2000兲.
Several cases of co-localisation between RGAs and
loci coding for major resistance genes have been ob-
served. These genes correspond generally to a gene-
for-gene system rather than a quantitative resistance

Table 1. Description of degenerate primers used for amplification of resistance and defence gene analogs. The sequences are coded according
to the International Units of Biochemistry: N 共A, G, T or C兲, Y 共C or T兲, B 共G, T or C兲, R 共A or G兲, H 共A, or C兲, M 共A or C兲, W 共A or T兲,
S 共G or C兲, K 共G or T兲, D 共G, A or T兲.
Primer Specificity Primer name sequence
NBS RGPS GGNATGGGYGGBRTHGGYAARAC
RGHDAS2 CARMGCYAAWGGYAADCC
Kinase domain P3 TNGGNSANGGNGKNTTYGG
P2R ACNCCRAANGARTANACRTC
PR2 Glu-S RYNGGWGTWTGYTAYGG
Glu-AS CADCCRCTYTCNGAYAC
PR5 PR5-S AACAAYTGYCCRTACACCGT
PR5-AS GGATCATCTTGWGGRTARCTATA
model. Common localisations, however, were ob-
served between major or isolate specific genes and
QTL 共Caranta et al. 1997; Ritter et al. 1991; Geoffroy
et al. 2000兲, suggesting possible structural similarities
between genes involved in qualitative and quantita-
tive types of resistance. A PCR-based approach has
been developed for several plant species, for example
pepper 共Pflieger et al. 1999兲, common bean 共Geoffroy
et al. 2000 兲, rice 共Wang et al. 2001 兲, sunflower
共Gentebittel et al. 1998兲, and wheat 共Faris et al. 1999兲
to characterise loci involved in quantitative resis-
tance. Co-localisations between analogs of resistance
or defence genes and QTL for polygenic resistance
have been observed 共see review by Pflieger et al.
2001b兲.
The aim of this study was to isolate and map in co-
coa analogs of resistance and defence genes that had
previously been cloned in other species and to com-
pare their genome location with that of QTLs related
to resistance to Phytophthora already identified in
mapping populations of cocoa.
Materials and methods
Plant material and DNA extraction
PCR amplification of DNA was performed on several
cocoa genotypes with primer pairs used to isolate
analogs of each class of gene:
– in the search for resistance gene analogs 共RGAs兲
corresponding to the N and Pto gene classes, DNA
from several cocoa clones was amplified: Scavina
6 共SCA6兲, an Upper Amazon Forastero clone
known for its high level of resistance, and from
C172 and C142 clones, both hybrids between
213
References
Geffroy et al. 共1999兲
Vallad et al. 共2001兲
Pflieger 共2001a兲
Pflieger 共2001a兲
UPA402, an Upper Amazon Forastero and UF 676,
a Trinitario selected by the United Company in
Costa Rica.
– in the search for defence gene analogs 共DGAs兲
corresponding to PR-2 and PR-5 proteins, DNA
from Catongo, a Lower Amazon Forastero clone
moderately susceptible to Phytophthora was ampli-
fied.
The mapping population used previously to establish
the cocoa genome reference map 共Risterucci et al.
2000兲 was used to map the RGAs and DGAs. This
population is composed of 100 trees derived from a
cross between UPA402 and UF676.
DNA extraction was performed according to Ris-
terucci et al. 共2000兲
PCR amplifications and cloning
Four pairs of degenerate primers were used to amplify
cocoa DNA 共Table 1兲:
RGSP and RGHDAS2 primers 共Geffroy et al.
1999兲 were designed respectively in the P-loop NBS
and the hydrophobic domain which have conserved
motifs among the NBS-containing R genes such as
RPS2, N or L6 共Leister et al. 1996兲
P3 and P2R primers were designed in the serine
threonine kinase 共STK兲 regions of Pto, Pti1, Fen
genes of tomato, and MHK and APK1 genes of Ara-
bidopsis 共Vallad et al. 2001兲. P3 is designed in the
STK subdomain I and P2R is designed in the subdo-
main IX.
Glu-s/GluAS and PR5-S/PR5-AS were designed
respectively from 18 PR-2 genes and 10 PR-5 genes
belonging to the Solanaceae family 共Pflieger et al.
2001a兲.
214
Amplifications were performed in a MJ Research
PTC 100 Thermal cycler on 100 ␮l reaction mixtures
containing 40 ng of cocoa DNA, 0.2 mM dNTP mix,
1,5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl 共pH
8.3兲, 1 ␮M primer and 2 units of Taq DNA
polymerase 共Eurobio兲. The samples were denatured at
94 °C for 4 min and subjected to 32 repeats of the
following cycle: 94 °C for 45 s, 50 °C 共 P3/P2R兲,
52 °C 共 Glu-s/GluAS兲 or 55 °C 共 PR5-S/PR5-AS and
RGSP/ RGHDAS2兲 for 1 min and 72 °C for 1 min.
PCR products were separated on a 1% agarose gel
and visualised under UV light after staining with
ethidium bromide. Selected bands were excised and
purified, then cloned into the pGemT vector
共Promega兲 according to the supplier’s instructions.
Characterisation of cloned DNA fragments
For each genotype, 100 independent clones from PCR
products amplified by RGPS / RGHDAS2 and by P3/
P2R primer pairs were characterised by restriction
analyses using three enzymes 共DraI, RsaI, XbaI兲 and
classified according to their restriction patterns.
Representative clones of each type of restriction pat-
terns were chosen for sequencing. Double stranded
plasmid DNA from selected clones was sequenced by
Genome Express using M13 forward and Reverse
primers.
Clones isolated from PCR products amplified by
Glu-S/Glu-AS 共PR2兲 and PR5-S/PR5-AS 共PR5兲 were
directly sequenced.
Sequence analyses and homology searches
Searches for DNA sequence homology were made
using WUBLAST 2 共Washington University Basic
Local Alignment Search Tool, Version 2.0兲 provided
by EBI 共European Bioinformatics Institute兲 and using
the EMBL database. DNA sequences were translated
into amino acids using Transeq provided by EBI.
Multiple sequence alignments of amino acid se-
quences were carried out with the MegAlign proce-
dure of DNASTAR software using Clustal V method.
The multiple alignment algorithm was the one
described by Higgins and Sharp 共1989兲. Phylogenetic
trees were built using the Clustal method based on
progressive multiple alignments to calculate distances
based on amino acid substitutions, and applying the
neighbor-joining method 共Saitou and Nei 1987兲 for
tree construction.
Mapping of RGAs and DGAs
New microsatellite markers mapped on the reference
map:
The reference map 共Risterucci et al. 2000兲 was
previously established with 424 markers comprising
5 isozymes, 178 RFLPs, 30 RAPDs, 191 AFLPs and
20 microsatellites. A new set of microsatellite mark-
ers was mapped on the reference map to provide a
larger number of suitable anchor markers which fa-
cilitated the comparison of candidate genes and QTL
locations on maps obtained using different cocoa
progenies.
Cocoa microsatellites: 18 microsatellites were pro-
vided by the Nestlé group. These were named Cx or
Cax. Thirty new microsatellites were produced by
CIRAD according to procedures described in Lanaud
et al. 共1999兲. These were named mTcCIRx 共EMBL
accession numbers AJ271822 to AJ271827 and
AJ271942 to AJ271960兲.
Cotton microsatellites: microsatellites isolated
from cotton by B. Burr 共Brookhaven National Labo-
ratory, USA兲 were tested for their potential to reveal
polymorphism in T. cacao, a species belonging to the
same order 共Malvales兲 as cotton. 216 cotton micro-
satellites were screened on 5 cocoa clones 共UPA402,
UF676, T60/887, IFC2, IFC5兲.
Restriction Fragment Length Polymorphism (RFLP)
analyses
The probes corresponding to RGAs or DGAs were
hybridised on genomic DNA of both parents re-
stricted by five enzymes 共EcoRI, EcoRV, HindIII,
XbaI and BglII兲 to identify probe/enzyme combina-
tions revealing polymorphisms. Useful probes were
then hybridised on the mapping population according
to procedures described in Risterucci et al. 共2000兲.
Linkage analysis
RAPD markers located on the first map established
by Risterucci et al. 共2000兲 were removed. New mic-
rosatellites and RFLP probes corresponding to RGAs
and DGAs were associated with the markers that had
been located on the first map, and their segregation
was studied on 100 individuals.
Linkage analyses were performed using the soft-
ware Joinmap version 3.0 共Van Ooijen and Voorrips
2001兲. As both parents were heterozygous, individual
markers segregated in the progeny according to two
possible Mendelian arrangements, heterozygous for
one parent and homozygous for the other parent, or

heterozygous for both parents with 2, 3 or 4 possible
segregating alleles. The markers that were heterozy-
gous in both parents allowed the production of an in-
tegrated map from the two individual parental maps.
Linkage groups were identified using a LOD score of
5.0. The Kosambi mapping function was used to con-
vert recombination frequencies into map distances
共Kosambi 1944兲.
Results
PCR amplification with degenerate primers and
characterisation of cloned sequences
Sequences containing NBS :
After PCR amplification of genomic DNA from the 3
clones SCA6, C142 and C172 using the RGPS and
RGHDAS2 primers defined from the NBS-class gene
and cloning the PCR product, insert sizes ranging
from 220 bp to 800 bp were observed.
A total of 34 clones were sequenced. Among them,
11 different sequences exhibited similarity to resis-
tance genes I2, I2C-1 and I2C-2 isolated from Lyco-
persicum esculentum by Ori et al. 共1997兲 and Simons
et al. 共1998兲. The pairwise high score varied from 367
to 826 and the nucleotide identity of cocoa sequences
to these genes from 59 to 65%.
Of the 11 RGA sequences, 10 could be translated
into polypeptides uninterrupted by stop codons. One
RGA contained a single stop codon 共N172/F12兲.
Multiple alignments of the amino acid sequences
situated between the P-loop and the GLPLAL motifs
were performed with the most similar clone 共I2 from
tomato兲 and the N resistance gene from tobacco be-
longing to the same class of genes. A significant
similarity was observed between all sequences, par-
ticularly at the level of the three NBS motifs 共P-loop,
kinase-2, kinase-3a兲 共Figure 1兲.
The percentage of similarity between cocoa protein
sequences varied from 46% to 100%, with 49% simi-
larity between the I2 tomato gene and the most simi-
lar cocoa sequence 共NSCA6/H8兲.
A phylogenetic tree was constructed to evaluate the
relationships among the sequences. This revealed 4
main cocoa RGA groups 共separated at d ⬎ 15兲 共Fig-
ure 2兲.
Homologous SRK sequences:
Using P3 and P2R primers, defined in the STK do-
main of Pto class genes, PCR products were cloned
215
and insert sizes ranging from 350bp to 1422bp were
observed. Forty cloned cocoa DNA fragments were
sequenced. Fifteen of these had a size close to 560 bp
and exhibited nucleotide similarity to the tomato Pto
resistance gene with a pairwise high score varying
from 407 to 1234 and a nucleotide identity varying
from 58 to 71%.
No stop codon was encountered in the 15 subse-
quent amino acid cocoa sequences homologous to the
Pto gene.
Another fragment with a size of 1422 bp 共PT172/
B9兲, appeared homologous to a wheat rust resistance
gene LRK10 共Feuillet et al. 1997兲, with a nucleotide
pairwise high score of 1211 and a nucleotide identity
of 59%. When translated into amino acids, 884 bp of
this DNA sequence had no stop codon. This part of
the PT172/B9 sequence is homologous to the coding
sequence of the LRK10 gene whose end is situated 13
amino acids before the first stop codon encountered
in PT172/B9.
After translation, the overall similarity between
protein sequences situated between the I and IX STK
subdomains was compared after multiple alignments
of the cocoa sequences and of the most similar resis-
tance genes: Pto from tomato and LRK10 from wheat
共Figure 3兲. Several conserved regions could be
observed in all subdomains of the STK region. Com-
parisons among the cocoa protein sequences homolo-
gous to Pto showed percentage similarity of between
43% to 100%. When compared to the Pto gene, the
most similar sequence 共PTSCA6/B2兲 showed 69%
similarity.
One cocoa sequence, PT172/B9 appeared to be
different from the others and was more similar to the
LRK10 gene 共63.8 % similarity兲 than to the other co-
coa sequences homologous to Pto 共31 to 40% simi-
larity兲 or to Pto 共38%兲. This variation among the
cocoa sequences also appeared very clearly on the
phylogenetic tree 共Figure 4兲 on which several groups
of sequences were identified.
Five other cocoa DNA fragments showed homol-
ogy with several other genes containing a serine/
threonine kinase domain. In plants, protein kinases
are known to play an important role in self incompat-
ibility systems. In this work, we isolated 2 DNA frag-
ments from cocoa showing homology with the
S-locus receptor kinase gene 共SRK3兲 involved in the
self-incompatibility system of Brassica and that also
contained a STK domain 共Goring and Rothstein 1992;
Stein and Nasrallah 1993兲.
216

Figure 1. Alignement of deduced amino acid sequences of RGA obtained after amplification of cocoa DNA with degenerate primers defined
by Geffroy et al. 共1999兲 in the P-loop and GLPLAL motif of NBS-LRR type resistance genes, and of 2 other resistance genes belonging to
the same class of genes: tobacco N 共Witham et al. 1994兲 and tomato I2 gene 共Ori et al. 1997兲. Residues that match the I2 gene, the closest
known resistance gene to cocoa RGA, are shaded in black.

PR- related proteins:
Using Glu-S/Glu-AS and PR5-S/PR5-AS primer
pairs defined in PR-2 and PR-5 homologues, DNA
fragments of expected length 共550 bp for the PR-5
and 750 bp for the PR-2兲 were amplified and cloned.
Insert sizes close to 550bp and 750bp respectively,
were observed.
Eighteen DNA fragments cloned from cocoa were
sequenced. Among them, six showed homology to the
␤ 1-3 glucanase gene 共PR2兲 and three to the thauma-
tin gene 共PR5兲 isolated from different species. The
nucleotide pairwise high scores varied from 1095 to
1747 and the nucleotide identity from 64 to 80%.
The similarity between cocoa protein PR-2-like se-
quences varied from 38 to 99% and their similarity
with the Gossypium glucanase gene 共closest defence
gene, accession number Z68154兲 共Hudspeth et al. un-
publ.兲 varied from 64% to 70% 共Figure 5兲.
The three cocoa protein PR-5-like sequences
showed between 67% to 74% similarity with the He-
lianthus PR-5 protein 共closest defence gene, acces-
sion number AF364864兲 共Hu, personal communica-
tion兲 共Figure 6兲.
 217

Figure 2. Phylogenetic tree based on alignment of the deduced amino acid sequences of cocoa RGA and the NBS domain of 2 R genes of the
same class: tobacco N gene and tomato I2 gene. Phylogenetic trees were built with DNASTAR/Megalign, using the Clustal V method based
on progressive multiple alignments to calculate distances 共d兲 共based on amino acid substitutions兲 and applying the neighbor-joining method
for tree construction 共Saitou and Nei 1987兲. The letters 共A, B, C or D兲 indicate the type of RFLP profile, and the adjacent number corre-
sponds to the chromosome where the RGA is located.

Mapping of RGAs and DGAs pattern, mapped in 2 clusters located on chromosome

Microsatellites
Cocoa microsatellites: Thirty new microsatellites
produced by CIRAD and 18 microsatellites provided
by the Nestlé group could be mapped.
Cotton microsatellites: Among the 216 cotton mi-
crosatellites screened on 5 cocoa clones, 11 amplifed
clear DNA fragments on cocoa and 5 revealed poly-
morphism. Three of these microsatellites could be
mapped on the reference map 共BNL 448, BNL 3089,
BNL 3932兲.
In total, a new set of 51 microsatellite markers was
mapped on the reference map
RGAs/DGAs
The RGAs and DGAs were first hybridized as RFLP
probes onto the DNA of the parents of the mapping
population, restricted by 5 enzymes 共EcoRI, EcoRV,
HindIII, XbaI, BglII兲 to identify polymorphic en-
zyme/probe combinations. Southern hybridisations
showed that each sequence corresponded to a unique
or low copy number of these sequences in the
genome.
Several types of RFLP patterns, completely differ-
ent, completely identical or sharing common bands,
were identified for each class of RGAs 共Figures 2 and
4兲 or DGAs: Four different types of RFLP banding
pattern were found after hybridisation with 12 probes
homologous to the NBS class resistance genes 共Fig-
ure 2兲. For each type of pattern, both common or var-
iable bands were observed according to the probes.
Four probes, representing these 4 types of banding
7 and on chromosome 10 共Figure 7兲. The 4 different
types of RFLP banding pattern reflect the structure of
the proposed phylogenetic tree at d ⬎ 15 共Figure 2兲.
Nine different types of RFLP patterns were obtained
after hybridisation with 16 RGA homologous to the
Pto or LRK10 genes 共Figure 4兲. The probes homolo-
gous to the Pto gene mapped onto five chromosome
regions 共on chromosomes 1, 2, 4, 9 and 10兲. A cluster
of RGAs located on chromosome 4 comprised five
RGAs with different RFLP patterns 共A4, B4, C4, E4,
H4兲 corresponding to sequences with variable amino
acid sequences 共Figure 4兲: one RGA homologous to
the LRK10 gene and 4 others homologous to the Pto
gene. Eight DNA fragments homologous to PR-2 and
PR-5 genes were hybridized. Among them, only 3
probes homologous to PR-2, representing 3 different
RFLP patterns could be mapped: two distant PR-2
sequences mapped together on chromosome 5 within
an interval of 6 cM, suggesting a cluster of defence
genes in this chromosome region. The other mapped
on chromosome 9 . In total, 16 different RGAs and
DGAs loci could be mapped 共Figure 7兲. The final map
had a total length of 839 cM and comprised 459
markers including 5 isozymes, 176 RFLPs, 191
AFLPs, 71 microsatellites, 13 RGAs and 3 DGAs.
Co-localisations between RGAs, DGAs and QTL
regions for resistance to Phytophthora
common markers 共RFLP or microsatellites兲 located
on the reference map and on the maps established by
several authors to identify QTL for resistance to Phy-
tophthora were used to compare the locations of
218

Figure 3. Alignement of deduced amino acid sequences of cocoa RGA obtained after amplification of cocoa DNA with degenerate primers
defined in the serine threonine kinase 共STK兲 domains 共I and IX兲 of tomato and Arabidopsis resistance genes 共Vallad et al. 2001兲 and of 2
other resistance genes belonging to the same class of R genes and close to the cocoa RGA: the tomato Pto gene 共Lavelle et al. personal
communication , EMBL acc. Number AF220603兲 and the wheat LRK10 gene 共Feuillet et al. 1997兲. Residues that match the Pto gene, the
closest known resistance gene to most of this class of cocoa RGAs, are shaded in black.
 219

Figure 4. Phylogenetic tree based on alignment of the deduced amino acid sequences of cocoa RGA and the STK 共serine threonine kinase兲
domain of 2 R genes of the same class : the tomato Pto gene 共EMBL acc. Number AF220603兲 and the wheat LRK10 gene 共Feuillet et al.
1997兲. Phylogenetic trees were built with DNASTAR/Megalign,. using the Clustal V method based on progressive multiple alignments to
calculate distances 共d兲 共based on amino acid substitutions兲 and applying the neighbor-joining method 共Saitou and Nei 1987兲 for tree con-
struction. The letters 共A to I兲 indicate the type of RFLP profile revealed by the RGA probe, and the adjacent number corresponds to the
chromosome where the RGA is mapped.

Figure 5. Alignment of deduced amino acid sequences of cocoa DGA obtained after amplification of cocoa DNA with degenerate primers
defined in the conserved domain of PR2 共Figure 5兲 and PR5 共Figure 6兲 genes belonging to the Solanaceae family 共Pflieger et al. 2001a兲. For
each class of PR the alignment was made with the closest PR gene: Gossypium hirsutum ␤ 1-3 glucanase gene 共Hudspeth et al. personal
communication兲 for the PR-2 analogs, and Helianthus PR5-1 gene 共Hu et al. personal communication兲, for the PR-5 cocoa analogs.
220

Figure 6. Alignment of deduced amino acid sequences of cocoa DGA obtained after amplification of cocoa DNA with degenerate primers
defined in the conserved domain of PR2 共Figure 5兲 and PR5 共Figure 6兲 genes belonging to the Solanaceae family 共Pflieger et al. 2001a兲. For
each class of PR the alignment was made with the closest PR gene: Gossypium hirsutum ␤ 1-3 glucanase gene 共Hudspeth et al. personal
communication兲 for the PR-2 analogs, and Helianthus PR5-1 gene 共Hu et al ., personal communication兲, for the PR-5 cocoa analogs.

RGAs/DGAs and QTL. Since the genetic distances
were not exactly the same between the same markers
located on the different maps, a correspondance was
made between the two more distant common markers
of each map and the corresponding markers of the
reference map. Several chromosome regions gather
RGA/DGA and QTL 共Figure 8兲:
– This is the case for the cluster of RGAs located on
chromosome 4. In the same region of about 10 cM,
four QTLs for resistance to P. palmivora have also
been identified: one QTL explaining 13.2% of the
variability in resistance to P. palmivora evaluated
by a fruit test was identified at LOD 3.4 共Figure
8-4C兲 in a Forastero clone 共Catongo兲 by Crouzillat
et al. 共2000兲 studying a progeny located in Costa
Rica. Two other QTL for resistance to P. palmivora
located in the same region were identified in an-
other Forastero clone 共IMC78兲 and in a Trinitario
clone 共DR1兲 at LOD 7.4 共Figure 8-4B兲 and 2.5
共Figure 8-4A兲 respectively by Clément et al. 共2003兲
studying progenies located in Côte d’Ivoire. These
QTLs explained respectively 22.6% and 10.1% of
the variability in percentage of rotten pods ob-
served in the field on data cumulated over a six
year harvesting period. Another QTL, also located
in this region of chromosome 4 and explaining
8.1% of the variability in resistance evaluated by a
leaf test, was identified at LOD 2.2 共Figure 8-4D兲
by Motilal et al. 共2000兲 in a Forastero clone
共IMC57兲 used as parent of a progeny located in
Trinidad.
– Another region of 15 cM on chromosome 5 groups
together 2 DGAs 共PR2 analogs兲 and QTL for re-
sistance to three different species of Phytophthora
共P. palmivora, P. megakarya, P. capsici兲 evaluated
using a progeny issued from a Trinitario clone 共H兲
by a leaf test by Risterucci et al. 共2003兲. These
QTL were identified at LOD 2.9 to 3.9 and
explained between 7.5 and 12.4% of the variability
for resistance depending on the strain and species
of Phytophthora studied.
– Another case of colocation could be observed in
chromosome 7: indeed, two RGA containing NBS
and one QTL for resistance to Phytophthora palm-
ivora were mapped in a same region of 10 cM
共Lanaud et al. 1999兲. This QTL was identified at
LOD 2,5 and explained 10% of the variability for
this resistance trait.
Discussion
Thirty six cocoa sequence homologs to known resis-
tance or defence genes were isolated using a
PCR-based approach with consensus primers. It was
possible to map some of these RGAs and DGAs on
the linkage groups 1, 2, 4, 5, 7, 9 and 10 of the ref-
erence map.
Four clusters of RGAs or DGAs were identified: 2
clusters of RGAs containing the NBS domain were
identified on chromosome 7 and 10. Five other Pto
and LRK10 analogs were also found, clustered in the
same region of chromosome 4. Out of the three
mapped DGAs homologous to PR-2, two were
located in the same region of chromosome 5.
Several RFLP patterns showed a clear and simple
locus pattern with only one or two bands correspond-

ing to homozygous or heterozygous individuals, and
could be mapped unambiguously. However some
other patterns revealed a few number of bands that
could correspond to different chromosome locations.
Indeed, due to the homology of the resistance gene
sequences, organised in clusters, and to the random
priming 32P labelling method used for RFLP analy-
ses, some specific sequences could appear mapped in
several chromosome regions. Recombination events
during PCR amplification steps of these homologous
sequences could also be responsible for some artifacts
during mapping. A BAC library recently constructed
共Clément et al. person. comm.兲 allowed the establish-
ment of the physical map of these RGAs/DGAs and
to compare this with their genetic maps.
Three major co-localisations could be considered
between the RGAs or DGAs isolated in this study and
QTL for resistance to Phytophthora. Indeed, the size
of the populations used for QTL analyses was not
generally higher than 150 or 200 individuals and the
QTL were not very precisely located, with a
confidence interval of about 15 to 20 cM. Fine map-
ping studies with larger populations would enable the
identified co-localisations to be refined.
A large number of resistance and defence genes
exist in plant genomes. In cocoa, 46 EST showing
similarity with known defence-associated genes were
recently isolated 共Jones et al. 2002兲. Seventy four
NBS containing sequences, organised in 11 catego-
ries, were also recently isolated from seven cocoa
clones by Kuhn et al. 共2003兲. In rice, about 800 EST
showing significant similarity to cloned disease resis-
tance and defence response genes were identified in
the Dupont rice EST database and 109 mapped on the
rice genetic map. They were sometimes associated
with QTL for resistance 共Wang et al. 2001兲. Due to
this high number of candidate genes spread across al-
most all chromosome regions, the probability of find-
ing a co-localisation between a QTL and a candidate
gene is high and many false associations could be re-
vealed. In order to confirm the hypothetical role of
these candidate genes in cocoa resistance, it is first
necessary to examine their level of expression during
infection. Transformation of cocoa has been recently
reported 共Maximova et al. 2003兲 and experiments on
transgene complementation would be the final step in
validating the role of the candidate genes and of the
several members of the cluster genes of resistance, as
has been done in tomato by Simons et al. 共1998兲 for
the dissection of locus I2C.
221
Among the RGA identified, the cluster situated on
chromosome 4 and located in the same region of
about 10 cM as four QTL of Phytophthora resistance
identified in various cocoa clones may contain seri-
ous candidates for explaining part of cocoa resistance
to Phytophthora.
Clusters of resistance or defence genes have been
previously reported in many other species 共Botella et
al. 1997; Faris et al. 1999兲 and our results indicate
that a similar organisation of resistance or defence
genes in clusters may also be encountered in cocoa.
This type of organisation in clusters is complex. In-
deed, it is known that a particular cluster could con-
tain both active genes and non-active or pseudo
genes. This situation has been reported in tomato for
the Pto-Fen cluster 共Martin et al. 1994兲 and the Mi
cluster 共Milligan et al. 1998兲. Moreover, gene mem-
bers of a particular cluster can confer different levels
of resistance. This is the case for locus I2 of tomato
that contains at least seven members belonging to the
NBS-LRR class of genes. One member 共I2C-1兲 con-
fers partial resistance to Fusarium oxysporum lyco-
persici race 2, while another 共I2C-K兲 confers total
resistance to this race 共Ori et al. 1997; Simmons et al.
1998兲. Sela-Buurlage et al. 共2001兲 suggested that
combined expression of more than one member could
be required to provide full field resistance. It was also
shown that a gene cluster can contain resistance genes
to several different pathogens. This is the case for to-
mato where one 1Mb region of chromosome 6 groups
together genes for resistance or tolerance to Cla-
dosporium fulvum 共Dixon et al. 1996兲, Oidium lyco-
persicum 共van de Beek et al. 1994兲 and TYLC virus
共Zamir et al. 1994兲. In potato, some resistance
hotspots grouping together QTL for resistance to dif-
ferent pathogens have also been observed 共Gebhardt
and Valkonen 2001兲.
In cocoa, several QTL involved in resistance to
different species of Phytophthora have been identified
in Scavina 6 and in the Trinitario clone H 共Risterucci
et al. 2003兲. Scavina 6 is a cocoa clone known for its
high resistance to other severe diseases such as
Witches’ Broom due to Crinipellis perniciosa and
Vascular Streak Dieback due to Oncobasidium theo-
bromae. A more precise analysis of gene clusters as-
sociated with QTL mapping studies is necessary to
understand the organisation of resistance genes in the
cocoa genome. A BAC library has been constructed
recently at CIRAD 共Clément et al. person. comm.兲
and will be a valuable tool for analysing the fine
222
 223

Figure 7. Genetic mapping of RGAs and DGAs isolated in the present study. The map has a total length of 839 cM and comprises 459 loci
including 5 isozymes, 176 RFLPs, 191 AFLPs, 71 microsatellites, 13 RGAs and 3 DGAs.
224

Figure 8. Comparisons between QTL 共ellipses兲 and RGAs/DGAs 共rectangles兲 mapping positions using common markers located on the link-
age groups 共LG兲 4, 5 and 7 of different maps 共the reference map 共REF兲 and the maps established by several authors to identify QTL for
resistance to Phytophthora兲: LG 4A and 4B 共Clément et al. 2003兲, LG 4C 共Crouzillat et al. 2000兲, LG 4D 共Motilal et al. 2000兲, LG 5 共Ris-
terucci et al. 2003兲 and LG 7 共Lanaud et al. 1999兲. Since the genetic distances were not exactly the same between the same markers located
on the different maps, a matching was established between the two most distant common markers of each map and the corresponding mark-
ers of the reference map. The name of the cocoa clone for which the QTL has been identified is reported for each QTL.

structure of the clusters of RGAs and DGAs in co-
coa.
Cocoa is affected by many other serious diseases,
and the RGAs and DGAs, already identified in this
study, may prove useful in defining the regions con-
ferring resistance to these diseases. Using classical
breeding strategies, the improvement of quantitative
resistance is difficult and time consuming. The iden-
tification of genes that control this quantitative resis-
tance would be particularly useful in the future to
provide markers more precisely mapped than QTLs
to control resistance gene introgressions during
breeding steps and to facilitate screening for new re-
sistance alleles in germplasm. The candidate gene ap-
proach developped in this work, combined with QTL
mapping strategies, constitutes a first step towards
defining the molecular basis of QTL for cocoa patho-
gen resistance. The enriched genetic map produced
with microsatellite markers in the present work and
especially those that are tightly linked to clusters of
RGAs or DGAs associated with QTL could help im-
prove resistance to pathogens in cocoa using marker
assisted selection strategies.
Acknowledgements
We thank CAOBISCO 共Association of Chocolate,
Biscuit and Confectionery Industries of the EU兲 for
their financial contribution to this work, and the CNS
共Centre National de séquençage兲 for the sequencing
of cocoa DNA fragments used in this work to produce
microsatellite markers. We are grateful to Brigitte
Courtois, Olivier Sounigo and Pietro Piffanelli for
useful comments on this paper.

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