Towards the understanding of the cocoa transcriptome: Production and analysis of an exhaustive dataset of ESTs of Theobroma cacao L. generated from various tissues and under various conditions.

Abstract

Theobroma cacao L., is a tree originated from the tropical rainforest of South America. It is one of the major cash crops for many tropical countries. T. cacao is mainly produced on smallholdings, providing resources for 14 million farmers. Disease resistance and T. cacao quality improvement are two important challenges for all actors of cocoa and chocolate production. T. cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses and quality improvement, nutritional and organoleptic, is also important for consumers. An international collaboration was formed to develop an EST genomic resource database for cacao.
Fifty-six cDNA libraries were constructed from different organs, different genotypes and different environmental conditions. A total of 149,650 valid EST sequences were generated corresponding to 48,594 unigenes, 12,692 contigs and 35,902 singletons. A total of 29,849 unigenes shared significant homology with public sequences from other species.Gene Ontology (GO) annotation was applied to distribute the ESTs among the main GO categories.A specific information system (ESTtik) was constructed to process, store and manage this EST collection allowing the user to query a database.To check the representativeness of our EST collection, we looked for the genes known to be involved in two different metabolic pathways extensively studied in other plant species and important for T. cacao qualities: the flavonoid and the terpene pathways. Most of the enzymes described in other crops for these two metabolic pathways were found in our EST collection.A large collection of new genetic markers was provided by this ESTs collection.
This EST collection displays a good representation of the T. cacao transcriptome, suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from these ESTs. It will provide numerous genetic markers that will allow the construction of a high density gene map of T. cacao. This EST collection represents a unique and important molecular resource for T. cacao study and improvement, facilitating the discovery of candidate genes for important T. cacao trait variation.

Full text

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BioMed Central
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BMC Genomics
Open Access
Research article
Towards the understanding of the cocoa transcriptome: Production
and analysis of an exhaustive dataset of ESTs of Theobroma cacao L.
generated from various tissues and under various conditions
Xavier Argout*1, Olivier Fouet1, Patrick Wincker2, Karina Gramacho3,
Thierry Legavre1, Xavier Sabau1, Ange Marie Risterucci1, Corinne Da Silva2,
Julio Cascardo4, Mathilde Allegre1, David Kuhn5, Joseph Verica6,
Brigitte Courtois1, Gaston Loor7, Regis Babin8,9, Olivier Sounigo8,9,
Michel Ducamp10, Mark J Guiltinan6, Manuel Ruiz1, Laurence Alemanno11,
Regina Machado12, Wilberth Phillips13, Ray Schnell5, Martin Gilmour14,
Eric Rosenquist15, David Butler16, Siela Maximova6 and Claire Lanaud1
Address: 1Biological Systems Department – UMR DAP TA 40/03, CIRAD, Montpellier, France, 2GENOSCOPE, 2 rue Gaston Crémieux, 91057 Evry,
France, 3CEPLAC, Km 22 Rod. Ilheus Itabuna, Cx. postal 07, Itabuna 45600-00, Bahia, Brazil, 4Laboratório de Genômica e Expressão
GênicaRodovia Ilhéus-Itabuna, UESC, Km 16, Ilhéus, Brazil, 5USDA-ARS, 13601 Old Cutler Rd. Miami, Florida, USA, 6Department of
Horticulture, The Pennsylvania State University, 422 Life Sciences Building, University Park, PA, 16802, USA, 7EET-Pichilingue, INIAP, Código
Postal 24 Km 5 vía Quevedo El Empalme, Ecuador, 8IRAD, Nkolbisson, BP 2067, Yaoundé, Cameroon, 9UPR 31 TA 80/02, CIRAD, Montpellier,
France, 10UMR BGPI TA41/K, CIRAD- 34398 Montpellier France, 11UMR BEPC TA 80/03, CIRAD, Montpellier, France, 12MASTERFOODS,
Almirante, Brazil, 13CATIE, P.O.Box 7170, Turrialba, Costa Rica, 14Mars Inc., Dundee Road, Slough, SL1 4JX, UK, 15National Program Staff, USDA-
ARS, Beltsville, Maryland 20705, USA and 16Cocoa Research Unit, The University of the West Indies, St. Augustine, Trinidad and Tobago
Email: Xavier Argout* - xavier.argout@cirad.fr; Olivier Fouet - olivier.fouet@cirad.fr; Patrick Wincker - pwincker@genoscope.cns.fr;
Karina Gramacho - karina@cepec.gov.br; Thierry Legavre - thierry.legavre@cirad.fr; Xavier Sabau - xavier.sabau@cirad.fr;
Ange Marie Risterucci - ange-marie.risterucci@cirad.fr; Corinne Da Silva - dasilva@genoscope.cns.fr; Julio Cascardo - cascardo@labbi.uesc.br;
Mathilde Allegre - maallegre@yahoo.fr; David Kuhn - David.Kuhn@ARS.USDA.GOV; Joseph Verica - joeverica@yahoo.com;
Brigitte Courtois - brigitte.courtois@cirad.fr; Gaston Loor - reyloor@yahoo.es; Regis Babin - regis.babin@cirad.fr;
Olivier Sounigo - olivier.sounigo@cirad.fr; Michel Ducamp - michel.ducamp@cirad.fr; Mark J Guiltinan - mjg9@psu.edu;
Manuel Ruiz - manuel.ruiz@cirad.fr; Laurence Alemanno - laurence.alemanno@orange.fr; Regina Machado - regina.machado@effem.com;
Wilberth Phillips - wphillip@catie.ac.cr; Ray Schnell - rschnell@ars-grin.gov; Martin Gilmour - martin.gilmour@eu.effem.com;
Eric Rosenquist - Eric.Rosenquist@ARS.USDA.GOV; David Butler - dbutler@intrepidequipment.com; Siela Maximova - snm104@psu.edu;
Claire Lanaud - claire.lanaud@cirad.fr
* Corresponding author
Abstract
Background: Theobroma cacao L., is a tree originated from the tropical rainforest of South
America. It is one of the major cash crops for many tropical countries. T. cacao is mainly produced
on smallholdings, providing resources for 14 million farmers. Disease resistance and T. cacao quality
improvement are two important challenges for all actors of cocoa and chocolate production. T.
cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses
and quality improvement, nutritional and organoleptic, is also important for consumers. An
international collaboration was formed to develop an EST genomic resource database for cacao.
Published: 30 October 2008
BMC Genomics 2008, 9:512doi:10.1186/1471-2164-9-512
Received: 4 June 2008
Accepted: 30 October 2008
This article is available from: http://www.biomedcentral.com/1471-2164/9/512
© 2008 Argout et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Results: Fifty-six cDNA libraries were constructed from different organs, different genotypes and
different environmental conditions. A total of 149,650 valid EST sequences were generated
corresponding to 48,594 unigenes, 12,692 contigs and 35,902 singletons. A total of 29,849 unigenes
shared significant homology with public sequences from other species.
Gene Ontology (GO) annotation was applied to distribute the ESTs among the main GO
categories.
A specific information system (ESTtik) was constructed to process, store and manage this EST
collection allowing the user to query a database.
To check the representativeness of our EST collection, we looked for the genes known to be
involved in two different metabolic pathways extensively studied in other plant species and
important for T. cacao qualities: the flavonoid and the terpene pathways. Most of the enzymes
described in other crops for these two metabolic pathways were found in our EST collection.
A large collection of new genetic markers was provided by this ESTs collection.
Conclusion: This EST collection displays a good representation of the T. cacao transcriptome,
suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from
these ESTs. It will provide numerous genetic markers that will allow the construction of a high
density gene map of T. cacao. This EST collection represents a unique and important molecular
resource for T. cacao study and improvement, facilitating the discovery of candidate genes for
important T. cacao trait variation.
Background
Theobroma cacao is a diploid species (2n = 2X = 20) with a
small genome size of 380 Mbp [1,2]. It is a tree fruit orig-
inating from the tropical rainforest of South America.
According to Cheesman (1944) [3], its center of origin is
the lower eastern equatorial slopes of the Andes. T. cacao
is now cultivated in all tropical lowlands of the world and
its beans are used to produce chocolate and cocoa butter
after a post harvest treatment including fermentation, dry-
ing and torrefaction steps. T. cacao is one of the major cash
crops for several tropical countries. Its economic impor-
tance is high and presently cocoa is the third most impor-
tant internationally traded raw material after sugar and
coffee.
Cocoa is mainly produced on smallholdings. It is esti-
mated that approximately 14 million people around the
world rely on cacao plantations for income. T. cacao pro-
duction is seriously affected by several fungal diseases and
insect attacks. Oomycetes and especially Phytophthora,
spp., (black pod) are responsible, worldwide, for 30% of
losses. Several species are involved. P. palmivora is present
in the entire cacao growing area, whereas P. capsici and P.
citrophthora are prevalent in South America. P. megakarya
is limited to some countries in West Africa, however it is
by far the most aggressive species causing losses of pro-
duction up to 50% Harvest losses due to Phytophthora spe-
cies were estimated to be 450,000 tons [4].
Two basidiomycetes, Moniliophthora roreri (frosty pod)
and Moniliophthora perniciosa (witches' broom) are also
responsible for important harvest losses. In Brazil, M. per-
niciosa was responsible for a drastic yield loss with a fall in
production from 405,000 tons in 1986 to less than
130,000 tons in 1998. Moniliophthora roreri causes a very
destructive pod rot and has already had dramatic effects in
some countries such as Ecuador [5] and Costa Rica [6]. M.
roreri was confined to several countries of Central and
northern South America, but is continuously spreading
towards other Central American countries like Mexico or
southward towards countries like Peru.
Several sources of disease resistance have been identified
in different genetic backgrounds, and the search for a sus-
tainable disease resistance, cumulating the different resist-
ance genes is one of the major challenges of T. cacao
genetic breeding programs [7].
Other traits of importance in T. cacao are quality traits.
Food quality improvement, nutritional as well as orga-
noleptic, is now a strong demand of consumers. Funda-
mental knowledge of the genetic basis of quality is an
important challenge that can address this demand.
Flavor is among the main criteria of quality for chocolate
manufacturers, but these characteristics are largely under-
studied by the cocoa research and breeding community
due to their complexity and a dramatic lack of fundamen-
tal knowledge about these traits. Flavour components
depend strongly on conditions of post-harvest processing
[8]. After pod harvests, fresh seeds need to be fermented
for 4 to 6 days, then dried and roasted to develope good

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cocoa aromas. Raw seeds, embedded in a pulp rich in
sugar, undergo biochemical changes under the effect of
various microorganisms present in the environment. The
initial anaerobic, low pH and high sugar conditions of the
pulp favour yeast activity, converting sugars in the pulp to
alcohol and carbon dioxide. Bacteria then start oxidising
the alcohol into lactic acid and then into acetic acid as
conditions become more aerobic. These biochemical
changes are accompanied by changes of amount and com-
position of several compounds having a major effect on
cocoa flavor such as peptide aroma precursor formation,
procyanidines or terpenes content.
However, it is now well recognized that the genetic origin
is also a strong determinant of flavor, independent of the
conditions of post-harvest processing [9].
Although some aromas are prominently defined by a sin-
gle molecule, most aromas are composed of a bulk of vol-
atile compounds responsible for aroma perception, and
belonging to different classes of organic compounds.
Interestingly, despite the vast number of chemical struc-
tures involved, the large majority of scent compounds are
biosynthesized by a surprisingly small number of meta-
bolic pathways. Parts of these metabolic pathways are
ubiquitous, and have been developed by small but impor-
tant modifications of ancestral genes and pathways [10].
In T. cacao more than 500 volatile compounds have been
detected. However, only a small number are thought to
play a key role in natural aroma variations.
Cocoa is classified into two classes: the «standard quality
cocoa» corresponding to 95% of the total market, and the
«fine flavor cocoa» produced by T. cacao trees originated
from two main varieties: Criollo and Nacional, which
bring a higher price in the market.
An important class of volatile compounds, the terpenes,
plays an important role in the aromatic flavor of these
varieties.
For example, a high level of linalool, a monoterpene, has
been observed in Nacional varieties [11] from Ecuador,
characterised by a floral taste, and could be at the origin of
this specific flavor which represents an important eco-
nomic «niche» for the country. However, the modern and
hybrid Nacional varieties present a wide range of flavor
variations due to introgressions of foreign and more vig-
orous varieties, leading to a dilution of this specific floral
flavor, and recently a part of Ecuador cocoa production
was declassified from fine flavor to "bulk cocoa" with a
lower price. An increased knowledge of the metabolic
pathways and expression of genes involved in terpene syn-
thesis could help to improve the aromatic flavor of new
"Nacional" varieties.
Independent to volatile compounds, some other bio-
chemical compounds are known to interact with T. cacao
organoleptic traits. This is the case with polyphenols. Cat-
echin, epicatechin and procyanidines are the main
polyphenols present in T. cacao. They have well known
antioxidant biological activities and beneficial effects on
the cardiovascular system [12-14]. Contributing to bitter-
ness and astringency, polyphenols influence T. cacao orga-
noleptic quality [15,12]. They influence aromatic profiles
of T. cacao in restricting Maillard's reactions, which gener-
ates a majority of the aromatic compounds of T. cacao.
Genomic research provides new tools to study the genetic
and molecular bases of important trait variations: EST
sequencing projects carried out on other plant models
have allowed the characterization of the transcriptome
and facilitated the gene discovery of important trait varia-
tions [16]. In tree crops, except for poplar whose genome
has been recently sequenced [17], genomic resources are
generally limited, and few large EST collections have been
produced. Recently, a citrus EST collection comprising
15,664 putative transcription units [18] has been pro-
duced, allowing the identification of clusters associated
with fruit quality, production and salinity tolerance. A
cotton study identified 51,107 unigenes from a global
assembly of 185,000 cotton ESTs, [19] providing a frame-
work for future investigation of cotton genomics. The
same approach was used to characterize the grape tran-
scriptome during berry development by the analysis and
annotation of 25,746 unigenes from 146,075 ESTs [20].
In T. cacao, only small collections of ESTs have been pro-
duced so far and used to study gene expression related to
stress or disease resistance and defense [21-24]
The objective of this study was to produce a large T. cacao
EST collection from a wide range of organs, providing a
good representation of T. cacao genes expressed during T.
cacao development and suitable for further analysis of all
kind of traits in T. cacao. Moreover, we emphasized the
production of tools to further study T. cacao diseases, a
major constraint for cocoa production, and quality fea-
tures. Therefore, we also produced cDNA libraries relevant
to disease resistance and quality traits. ESTs were pro-
duced from T. cacao tissues interacting with various pest
and fungal diseases, from seeds at different stages of devel-
opment and during the fermentation steps. This large EST
collection will provide valuable tools to carry out func-
tional genomic studies and discover genes essential to
important agronomic and quality trait variation in T.
cacao, aiming to accelerate T. cacao improvement. A multi-
disciplinary approach combining functional genomic and
quantitative genetic approaches could lead to a better
understanding of gene function involved in disease resist-
ance mechanisms or quality trait variations. T. cacao's phy-

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logenetic proximity to the model plant Arabidopsis will
facilitate our understanding of most metabolic pathways.
However, T. cacao is a tree, and expresses traits not found
in Arabidopsis, thus we hypothesize that genes not found
in Arabidopsis play important roles in cacao development.
Results and Discussion
Library construction
Fifty-six libraries were constructed from two main geno-
types representing three contrasting genetic origins: ICS1,
a hybrid between Criollo and Forastero from Lower Ama-
zonia of Brazil, and Scavina 6, a Forastero from Upper
Amazonia of Peru. A few other genotypes characterized by
specific resistance or quality traits and belonging to vari-
ous genetic origins were also used. The plant materials
were provided from a various panel of different T. cacao L.
organs (Table 1). Among them, 25 libraries corresponded
to T. cacao tissues introduced to different biotic stresses:
pods inoculated by Phytophthora palmivora, Phytophthora
megakarya, Moniliophthora perniciosa and Moniliophthora
roreri, leaves inoculated by Phytophthora palmivora and
Phytophthora megakarya, stems inoculated by Monilioph-
thora perniciosa and Ceratocystis fimbriata, and stems
attacked by Sahlbergella singularis (mirids). Among these
libraries, 17 are suppressive subtractive hybridization
(SSH) libraries. Finally, two libraries corresponded to T.
cacao tissues introduced to drought stresses and 11 corre-
sponded to seed development and fermentation stages.
EST sequencing and assembly
From the 56 libraries, 8565 clones were first sequenced on
both strands using forward and reverse primers, to have
an overview of the quality of the libraries, and then
163,868 clones were single-pass sequenced from 5' or
from 3' end (Table 1). This represented a total number of
180,998 chromatograms that were used in this analysis.
After low quality, vector and adapters trimming, 149,650
sequences longer than 100 bp remained as good quality
sequences. The average sequence length was 472 bp and
62% were longer than 400 bp. These individual ESTs
(available through EMBL-Bank [25]) were assembled
using the TIGR Gene Indices clustering tools (TGICL)
[26]. The assembly process produced 12,692 contigs and
35,902 singletons that represented a total of 25.6 Mb of
transcripted sequences. The combined set of contigs and
singletons resulted in 48,594 unigenes which might corre-
spond to different putative transcripts or different parts of
the same transcript found in the Theobroma cacao tran-
scriptome. The average length of this T. cacao non redun-
dant sequences dataset was 527 bp.
An assembly of ESTs has already been published for Theo-
broma cacao but has been limited to 1380 unigenes (4433
ESTs) from two leaf and bean cacao libraries [21], to the
isolation of 1256 unigenes (2114 ESTs) from cacao leaves
treated with inducers of defense response [23] and to
2926 non redundant sequences from libraries of cacao
meristems inoculated by Moniliophthora perniciosa [24].
The results of this study are more comparable to a cotton
EST project [19], involving 30 cDNA libraries. This analy-
sis detected 51,107 unigenes in approximately 185,000
Gossypium ESTs.
Analysis of EST abundance in a contig can provide
insights to gene expression levels, although this informa-
tion must be taken with caution due to cloning and repli-
cation bias resulting form library construction and
propagation steps. The number of ESTs in the T. cacao con-
tigs ranged from 2 to 5102 (Figure 1) and 65.3% were
composed of 4 or less ESTs. 98% of the contigs contained
less than 50 ESTs.
We evaluated the redundancy of transcripts in each library
and among all libraries by studying the distribution of
ESTs in contigs across multiple libraries. 11,226 had
members from more than one library (Figure 2) and 1466
contigs were specific from one library. No contigs had
members from all 56 libraries. Two contigs were found in
52 libraries: the contig CL1Contig269 was similar to the
mitochondrial large subunit ribosomal RNA gene and the
contig CL1Contig513 to the 18S ribosomal RNA gene.
The contig CL18Contig2, CL2Contig3 and CL15Contig2,
similar to an ATP Synthase beta subunit, a metal-
lothionein-like protein and a photosystem II D1 protein
respectively, were found in 47 libraries.
Unigene set annotation
BLASTN against cacao ESTs
The unigene dataset was used to detect how many cacao
sequences had not been already described in public data-
bases. To answer this question, we collected all 2539 T.
cacao unique sequences already published by the Dana
Farber Cancer Institute (DFCI) gene index [27] and we did
a BLASTN search against our unigenes. An e-value cutoff
of 1e-50 was used to ensure that only highly similar
sequences were detected. A total of 3901 unigenes pro-
duced a significant hit with 1788 unique sequences from
the DFCI gene index, therefore these sequences may cor-
respond to T. cacao sequences already published or may
match different parts of the gene index sequences. They
may be also produced by closely related genes (multigenic
families). Finally, 44,693 unique sequences did not pro-
duce a significant hit, therefore these sequences may be
new.
BLASTX and BLASTN annotation
The unigenes were first translated into amino-acid
sequences and then searched for similar protein with the
BLASTX program using an e-value cutoff of 1e-5 against

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Table 1: Summary of T. cacao libraries
Genotype LibraryLibrary descriptionGood quality ESTsUnigenes
Jaca
Scavina6
CERATOJ_KZ0ACI
CHERELS_KZ0AAC
stem tissues inoculated by Ceratocystis fimbriata
cherels from 1 week to 1 month stage of development
1729
4252
1270
2836
Scavina6
Scavina6
Scavina6
ICS1
COPHAS_KZ0AAL
CORTEXS_KZ0AAT
CORTINS_KZ0AAV
COSSHPPI_KZ0AA
pod tissue inoculated by Phytophthora palmivora
cortex tissue, external part
cortex tissue internal part with lignified chanels
SSH library from tissues inoculated/non inoculated by
Phytophthora palmivora
4905
3817
5096
1721
2621
2227
3331
955
Scavina6 COSSHPPS_KZ0AASSH library from tissues inoculated/non inoculated by
Phytophthora palmivora
cotyledons from germinated seeds (1 to 3 weeks)
young cushions
leaves submited to drought stresses
roots submited to drought stresses
epicotyle and hypocotyle from 1 week germinated seeds
17021129
ICS1
B97 C-C-2
Scavina6
Scavina6
ICS1AF
COTYLEI_KZ0ABB
CUSHIONC_KZ0ACAC
DROUGHTLS_KZ0ACAF
DROUGHTRS_KZ0ACAE
EMBR1WI_KZ0ABA
5153
2849
2766
2685
3246
2961
2120
1290
1563
2473
ICS1AF
Scavina6
Scavina6
EPIC23I_KZ0AAS
FLOWERS_KZ0AAD
FLPOLSSH_KZ0ABL_M
epicotyle from 2–3 week germinated seeds
flowers at different stages of development
SSH library from ovaries submitted to compatible/incompatible
pollinations
hypocotyle from 2–3 week germinated seeds
young and adult leaves at different stages of development
3005
3511
2398
2459
2434
431
ICS1AF
Scavina6
HYPO23I_KZ0AAP
LEAVES_KZ0ABE
5111
4698
2955
3069
GU255V
PNG seedlings
PNG seedlings
LEAVPAGU_KZ0ACQ
LEPAPNGR_KZ0ACP
LESSHMEPNGa_KZ0ACAP
leaves inoculated by Phytophthora palmivora
leaves inoculated by Phytophthora palmivora
SSH library from leaves inoculated by Phytophthora megakarya
from susceptible-resistant PNG seedlings
SSH library from leaves inoculated by Phytophthora megakarya
from resistant – susceptible PNG seedlings
3030
1021
356
2139
862
169
PNG seedlingsLESSHMEPNGb_KZ0ACV1244 749
PNG seedlingsLESSHPNGRSb_KZ0ABPSSH library from leaves inoculated by Phytophthora palmivora
from resistant – susceptible PNG seedlings
young shoot tissues attacked by Sahlbergella singularis (mirids)
701 438
UF676MIRIDUFS_KZ0ACAD3011 1908
P7
UF273
IMC47
ICS1
UPA134
Scavina6
MONILIOP_KZ0AB
MONILIOU_KZ0ABV
OVUL1_7M_KZ0ACAK
OVULEI_KZ0AAB
PODMEUPA_KZ0ACAB
PODSSHWB1Sb_KZ0ACD
pod tissues inoculated by Moniliophthora roreri
pod tissues inoculated by Monilia roreri
ovaries from 1 to 7 days after pollinations
ovules collected 2 to 3 months after pollination
pod tissues inoculated by Phytophthora megakarya
SSH library from pod tissues inoculated-non inoculated by
Moniliophthora perniciosa less than 60 days after inoculation
3074
3159
1565
4942
3492
652
2217
1871
1218
3315
2093
534
Scavina6 PODSSHWB2Sb_KZ0ACFSSH library from pod tissues inoculated-non inoculated by
Moniliophthora perniciosa between 60 to 120 days after
inoculation
pod tissues inoculated by Moniliophthora perniciosa less than
60 days after inoculation
pod tissues inoculated by Moniliophthora perniciosa between
60 to 120 days after inoculation
SSH library from leaves of resistant seedlings inoculated-non
inoculated by Phytophthora megakarya
roots
SSH library from leaves of resistant seedlings non inoculated-
inoculated by Phytophthora palmivora
SSH library from leaves of resistant seedlings inoculated-non
inoculated by Phytophthora palmivora
seeds 3 to 3,5 months after pollinations
seeds 4 to 5 months after pollinations
Cotyledons from seeds fermented between 6 H and 4 days
1399912
Scavina6 PODWB1S_KZ0ACM17041213
Scavina6 PODWB2S_KZ0ACN1718 1217
PNG seedlings RESSHMEPNGb_KZ0AC1287 931
Scavina6
PNG seedlings
ROOTS_KZ0ABF
RPPSSHPNGa_KZ0ACAL
3567
344
2892
266
PNG seedlingsRPPSSHPNGb_KZ0ACR1407823
ICS1
ICS1
33–49
SEED34I_KZ0AAH
SEED45I_KZ0AAE_F
SEEDFERB_KZ0ACAG
3942
3296
1664
2637
1902
465
ICS1SEEDMAI_KZ0AAG seeds from mature pods 5,5 to 6 months after pollinations30681844

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the non-redundant protein sequence database (NR) with
entries from GenPept, Swissprot, PIR, PDF, PDB and
NCBI RefSeq. The 10 best hits were retained for the anno-
tation, providing an annotation for 27,245 cacao
sequences (56.1%). The 43.9% of the unigenes that did
not have any match were searched for similar nucleotide
sequences from the Genbank nucleotide collection NT
with the BLASTN program. An e-value cutoff of 1e-5 was
also used and the 10 best hits were used for the annota-
tion. 2604 unigenes exhibited a significant similarity with
nucleotide sequences providing a BLASTX or BLASTN
annotation for 29,849 unigenes. The 10 BLASTX hits were
used to classify the unigenes according to the species asso-
ciated with the annotation (Figure 3A). A total of 140,270
hits (56%) involved proteins from Vitis vinifera, Arabidop-
sis thaliana or Oryza sativa, while 1955 hits involved pro-
teins from Gossypium hirsutum, a closely related species
from the Malvaceae family [28]. Although fewer protein
sequences from Vitis vinifera than from Arabidopsis thaliana
(54,395 and 58,061 respectively) were present in the non
redundant database we used for BLASTX, and although
the evolutionary distance between Vitis vinifera and Theo-
broma cacao is higher than the distance between Arabidop-
sis thaliana and Theobroma cacao [28], we found more
similarities with Vitis vinifera (50,315 hits) than with Ara-
bidopsis thaliana (41,766 hits).
To further investigate this unexpected result we compared
with the BLASTX program the cacao unigenes dataset
against the two proteomes of Arabidopsis thaliana and Vitis
vinifera (Figures 3B, C). For each Blast result, we selected
the species found in the first hit having an expected value
lower than 1e-15 to detect similar sequence. A total of
25,049 Theobroma cacao sequences (56%) presented at
least a significant hit with an Arabidopsis thaliana or Vitis
vinifera protein. The results showed that 18,643 Theobroma
cacao sequences presented a higher similarity to the Vitis
vinifera proteome whereas only 6406 Theobroma cacao
sequences presented a first Blast hit similar to the Arabi-
dopsis thaliana proteome (Figure 3B). Moreover, it was
determined that these first significant hits involved 9943
Vitis vinifera proteins (33% of the proteome) and 4246
Arabidopsis thaliana proteins (12% of the proteome) (Fig-
ure 3C).
These surprising results suggest that the genes expressed in
Theobroma cacao are more similar to Vitis vinifera proteins
than to those of Arabidopsis thaliana. These findings could
be explained by the fact that Theobroma cacao and Vitis vin-
ifera are both fruit trees. This idea could be supported by
the large amount of Blast hits found with other tree crops
such as Populus trichocarpa (8605 Blast hits), despite a
small number of non redundant proteins in the databases
for this species.
Gene Ontology annotation
We used BLAST2GO [29], a program that retrieves GO
terms based on BLAST definition, to assign gene ontology
BE240
ICS1
ICS1
Jaca
SEEDNAB_KZ0ABH
SEFERMI_A_KZ0AAR
SEFERMI_B_KZ0AAM
SSHCERATOJb_KZ0ACS
seeds 2 to 5 months after pollinations
fermented seeds during 6 to 26 H
fermented seeds during 32 to 40 H
SSH library from stems inoculated-non inoculated by
Ceratocystis fimbriata
SSH library from stems non inoculated-inoculated by
Ceratocystis fimbriata
SSH library from young shoots non attacked-attacked by
Sahlbergella singularis
SSH library from young shoots attacked-non attacked by
Sahlbergella singularis
complete disc of stems 1 cm diameter
SSH library from (and reverse sens) shoot tissues inoculated/
non inoculated by Moniliophthora perniciosa less than 18 days
after inoculation
4988
1798
3931
339
3101
844
2110
327
Jaca SSHCERATOJa_KZ0ACAM 1364 918
UF676SSHMIRUFa_KZ0ACAN 320 296
UF676SSHMIRUFb_KZ0ACT13931051
Scavina6
Scavina6
STEMS_KZ0AAA
STSSHWB1S_KZ0ABI_K
4938
1594
2880
370
Scavina6STSSHWB2Sb_KZ0ACBSSH library from shoot tissues inoculated-non inoculated by
Moniliophthora perniciosa between 18 to 120 days after
inoculation
14081056
33–49
ICS1
Scavina6
TEGFERB_KZ0ACAH
TEGPULI_KZ0AAI_K
TISCIVS_KZ0AAQ
testa from seeds fermented between 6 H and 4 days
testa with pulp from mature seeds
embryogenic and non embryogenic callus in vitro culture
1649
5017
3434
808
3254
2389
ICS1
P7
Scavina6
TPFERMI_A_KZ0AAN
WILTP_KZ0ACL
WOODS_KZ0ACAA
fermented testa during 6 to 40 H
young wilted cherels 7 to 10 days after pollination
bark and cambium part of wood
4005
1706
3478
2164
1247
2234
Table 1: Summary of T. cacao libraries (Continued)

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(GO) annotation [30] to the unigene dataset. To best
exploit GO results, we built a local AmiGO browser [31].
A total number of 49,364 annotations were found and
16,364 unigenes were characterized by at least one anno-
tation. These annotations were distributed among the
main GO categories into 16,448 Biological Process (P),
14,696 Cellular Component (C) and 18,219 Molecular
Function (F) (Figure 4A). The most abundant high-level
direct GO counts within these categories were C: mito-
chondrion (1924), C: membrane (1218), C: plastid
(1173), F: ATP binding (1017) and C: chloroplast (1001)
(Figure 4B).
Genes involved in defense and resistance mechanisms
Some of the libraries provide an important resource to
study plant/pathogens interactions. Using the annota-
tions provided by Blast and Gene Ontology, we specifi-
cally focussed on genes known to play a crucial role in
plant pathogen resistance and defense mechanisms [32].
Using the AmiGO browser, we identified 1001 gene prod-
uct associations to "response to stress" (GO:0006950).
Both searches with Blast result and Gene Ontology anno-
tation resulted in the identification of unigenes similar to
known proteins involved in resistance or defense mecha-
nisms such as LRR-NBS [33] (8 contigs and 32 single-
tons), chitinase [34] (19 contigs and 37 singletons), 1–3
beta glucanase [35] (5 contigs and 7 singletons) or patho-
genesis-Related protein (24 contigs and 24 singletons).
Other genes related to resistance/defense mechanisms
were also found more specifically in libraries produced
from pathogen infected tissues, such as those involved in
regulation of pathogen-induced genes like transcription
factors (6 contigs and 7 singletons), in signal transduction
(like MAPKinase with 5 contigs and 3 singletons) or in the
cell death program.
The identification of a unigene set gathering sequences
from all genes known to be involved in plant resistance
and defense mechanisms, and the construction of a corre-
sponding microarray could constitute a valuable tool to
progress in the understanding of plant/pathogens interac-
tions.
Genes involved in particular metabolic pathways or
biological activities
To check the representativeness of our EST collection, we
looked for ESTs encoding proteins known to be involved
in the flavonoid and the terpene pathways, already stud-
ied in other plant species, and at the basis of important
Distribution of T. cacao EST members in contigs after the assembly process
Figure 1
Distribution of T. cacao EST members in contigs after the assembly process.

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traits of interest in T. cacao. Generally, polyphenols play a
major role in chocolate quality, acting as colour precur-
sors or taste agents [36]. Moreover, they are strongly
implicated in health benefits associated with chocolate
consumption [37-40].
The flavonoid pathway
The flavonoid pathway has been already studied in several
plants [41]. In T. cacao, this pathway is the source of
numerous essential components for human health bene-
fits of chocolate [37-40] and resistance against pathogens
[42].
Gene Ontology analysis highlighted 99 EST sequences
implicated in "phenylpropanoid biosynthetic process"
(GO:0009699), most of them implicated in flavonoid
biosynthesis. For example, the GO analysis, together with
keyword ESTtik database searching (see material and
methods) into Blast Results allowed us to find sequences
encoding phenylalanine ammonia lyase (5 contigs and 12
singletons), cinnamate-4-hydroxylase (4 contigs and 11
singletons), the 4-coumarate-CoA ligase (14 contigs and
12 singletons), chalcone synthase (6 contigs and 25 sin-
gletons) and chalcone isomerase (8 contigs and 13 single-
tons), all major enzymes of the general flavonoid pathway
(Figure 5). Most specific enzymes, implicated in anthocy-
anin biosynthesis (flavanone-3-hydroxylase, dihydrofla-
vonol reductase, anthocyanidin synthase, flavonoid-3-
glucosyltransferase) were also represented in this T. cacao
EST resource.
The terpene pathway
Terpenoid compounds, synthesized in the isoprenoid
pathway (Figure 6), are compounds of importance for
specific scent and aromatic qualities of chocolates classi-
fied as "fine and flavor". For example, linalool, a monot-
erpenol, is found in high quantity in Arriba Nacional
varieties from Ecuador and in some Criollo clones from
Venezuela [11,43,44]. Linalool, together with other vola-
tiles, could be responsible for the typical floral aroma [45]
of these chocolates.
One of our goals was to identify enzymes involved in the
terpenoid pathway that could be responsible for linalool
content variations among Nacional clones. As a first step
we identified sequences encoding isoprenoid pathway
enzymes (42 contigs and 55 singletons). The final step
enzyme for linalool synthesis, linalool synthase, was rep-
resented by 2 contigs and 4 singletons. Nearly all enzymes
reported to be involved in this biochemical pathway were
present in our ESTtik database, allowing the analysis of
the T. cacao terpene pathway based on oligonucleotide
microarrays derived from these ESTs.
The fact that nearly all of the genes involved in these two
pathways as described in other plant species were identi-
Number of contigs composed from sequence originated from one ore more libraries
Figure 2
Number of contigs composed from sequence originated from one ore more libraries.

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Species distribution among the Blast results of T. cacao unigenes
Figure 3
Species distribution among the Blast results of T. cacao unigenes. A – Distribution of species represented in the 10
first Blast hits against NCBI Non redundant protein database. B – Number of best Blast hits against Arabidopsis thaliana and Vitis
vinifera proteomes. C – Arabidopsis thaliana (black columns) and Vitis vinifera (grey columns) proteome coverage.

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Gene Ontology annotation results
Figure 4
Gene Ontology annotation results. A – Distribution of the unigenes among the main Gene Ontology categories (Biologi-
cal Process, Cellular Component and Molecular Function). B – Distribution of the unigenes among the 10 best Gene Ontology
terms.

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fied in ESTs from our collection demonstrates the high
level of representation of this resource and suggests that
the majority of cacao genes have been sampled. Thus, this
EST collection offers a comprehensive resource to search
for candidate genes involved in quality traits and other
important agronomical traits variation.
Production of SSR and SNP markers
Molecular markers derived from ESTs are part of, or adja-
cent to genes, and therefore they provide an efficient
means of gene mapping.
Simple Sequence Repeats (SSRs) were identified in the
unigene dataset with the MISA pipeline [46]. In this study,
SSRs were defined as dimers with at least 6 repetitions and
trimers, tetramers, pentamers and hexamers with at least
5 repeats. Microsatellites were considered compound
when two SSRs were not separated by more than 100 bp.
A total of 2252 SSRs were identified as 2164 unigenes, and
204 unigenes had more than 1 SSR. Dimers and trimers
were the most common types (Table 2) and represented
94.2% of SSRs found in unigenes. The distribution of all
possible dimer and trimer motifs found in the unigenes is
Schematic overview of the general flavonoid biosynthesis pathway (according to Schijlen et al., 2004; Marles et al., 2003)
Figure 5
Schematic overview of the general flavonoid biosynthesis pathway (according to Schijlen et al., 2004; Marles et
al., 2003). The number of contigs and singletons present in our EST dataset was added between brackets for each enzyme.

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Figure 6 (see legend on next page)

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listed in Table 3. The poly(AG)n and poly(AAG)n groups
were the most abundant motifs in T. cacao unigenes.
For each SSR identified, if possible, 3 couples of primers
were defined using Primer3 [47]. A total of 5265 flanking
sequences were designed and it was possible to define at
least one couple of primers for 1755 SSRs.
The exploration of redundant ESTs in contigs was shown
to be a valuable resource of Single Nucleotide Polymor-
phisms (SNP) [48]. SNPs were detected using QualitySNP
[49] pipeline from unigene contigs. We assumed that con-
tigs with at least 100 members contained paralogous
sequences [50,51] therefore we selected 4818 contigs that
contained at least 4 sequences but no more than 100
sequences. A preliminary study assembled 5246 SNPs into
2012 contigs. Transitions (A/T-G/C) represented 54.2% of
the SNPs found, transversions 32.1% and InDels 13.7%.
Conclusion
The present assembly of 149,650 T. cacao ESTs produced
from 56 cDNA libraries constructed from different organs
and environmental conditions is the largest transcriptome
dataset produced so far for T. cacao, and among the largest
ones generated for any tree fruit crop. It provides a major
resource for cacao genetic and functional genomic analy-
ses of important T. cacao traits, with the identification and
annotation of 48,594 different putative transcripts.
The improved knowledge of the T. cacao transcriptome
will enhance our understanding of main disease resistance
mechanisms and will be useful to improve new varieties
and establish a sustainable T. cacao resistance to pests and
diseases. Towards this goal, a large number of cDNA
libraries have been produced from T. cacao/pathogens or
pest interactions, and an important set of unique tran-
scripts homologous to genes known in other species
involved in defense and resistance mechanisms have been
identified in the whole EST collection using keywords and
Gene Ontology tools. It provides a cDNA resource availa-
ble for the broad scientific community and suitable for
cDNA-based microarray analyses.
This collection of ESTs also provides a valuable frame-
work for the discovery of candidate genes involved in
chocolate quality traits. Tested for two distinct metabolic
pathways, this collection displays a good representation
of the T. cacao transcriptome involved in quality trait elab-
oration and will allow the comparative analysis of con-
trasting genotypes for T. cacao qualities to better
understand the genetic basis of quality.
This EST collection also will provide a large number of
genetic tools, such as SSR and SNP markers, which will be
used to construct high density gene maps, facilitating the
integration of genetic and genomic approaches to dis-
cover the genes that effect trait variations, and also facili-
tating the sequence assembly in further activities of whole
T. cacao genome sequencing.
Finally, the assembly and annotation associated will also
provide a valuable resource for future investigation of T.
The biosynthesis pathway of isoprenoïdes
Figure 6 (see previous page)
The biosynthesis pathway of isoprenoïdes. (according Liu et al., 2005). Pathway Mevalonate (MVA) cytoplasmic in left and
pathway 1-deoxyxylulose-5-phosphate (DXP) chloroplastic in right. AACT, acetoacetyl-coenzyme A (CoA) thiolase; CMS, 2-
C-methyl-D-erythritol 4-phosphaate cytidyl transferase; DTS, diterpene synthase; DXR, 1- deoxy-D-xylulose 5-phosphate
reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; FPPS, farnesyl diphosphate synthase; GGPPS, geran-
ylgeranyl diphosphate synthase; GPPS, geranyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase; IPPi, isopentenyl diphosphate isomerase; MTS, monoterpene synthase; SES, sesquiterpene synthase;
SQS squalene synthase; MK, mevalonate kinase; MPK, mevalonate-5-phosphate kinase; CMK, 4-(cytidine 5'-diphospho)-2-C-
methyl-D-erythritol kinase; MDD, mevalonate diphosphate decarboxylase; IDS, isopentenyl diphosphate/dimethylallyl diphos-
phate synthase; MCS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, 1-hydroxy-2-methyl-2-(E)-butenyl 4-
diphosphate synthase; PSY, phytoene synthase; HMGS, HMG-CoA synthase; HMG-CoA, 3S-hydroxy-3-methylglutaryl coen-
zyme A; DXP, 1-deoxy-D-xylulose 5-phosphate; MVA, 3R-Mevalonic acid; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-
ME, 4-(cytidine 5'-diphospho)-2C-methyl-D-erythritol; CDP-MEP, 4-(cytidine 5'-diphospho)-2C-methyl-D-erythritol 2-phos-
phate; cMEPP, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; DMAPP, Dimethylallyl diphosphate; HMBPP, 1-hydroxy-2-
methyl-2-(E)-butenyl 4-diphosphate; IPP, isopentenyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate;
GGPPS, geranylgeranyl diphosphate. The number of contigs and singletons present in our EST dataset was added between
brackets for each enzyme.
Table 2: Distribution of motifs length in SSRs dataset
Motif Length Number of SSRsFrequency
2
3
4
5
6
compound
1132
857
82
35
14
132
50.3
38.1
3.6
1.6
0.6
5.9

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cacao evolutionary genomics with related species such as
Gossypium hirsutum or Arabidopsis thaliana.
Methods
Material used for libraries construction
In total, 56 different libraries were constructed. The
organs and T. cacao genotypes used for cDNA construc-
tion, and the treatments carried out on these organs are
reported in Table 1.
Most of the libraries were constructed from 2 genotypes:
- Scavina 6 (SCA6) is a self incompatible Forastero geno-
type originating from the Upper Amazonian region of
Peru. SCA6 is highly resistant to Phytophthora species and
Moniliophthora perniciosa diseases. It has been widely used
in the breeding programs.
- ICS1 is a self compatible Trinitario genotype, a hybrid
involving Criollo, the first T. cacao variety domesticated in
Central America, and a Forastero variety originated from
the Lower Amazonia of Brazil; ICS1 is known for its large
beans and good quality traits. This clone was used for
RNA production during the different stages of develop-
ment of the T. cacao seeds.
A post harvest treatment is generally applied to T. cacao
seeds to develop chocolate, involving fermentation steps,
drying and torrefaction. Tissues from ICS1 Seeds were col-
lected during the first 2 days of fermentation to construct
cDNA libraries.
Other genotypes were used more specifically to represent
particular traits or genetic origins:
- Jaca is a Brazilian Forastero genotype from the Upper
Amazonian region, and resistant to Ceratocystis fimbriata.
Inoculation was done according to Silva et al. [52]
- B97 C-C-2 is a pure and homozygous Criollo genotype.
This material was collected in Belize [53] by a mission
conducted by the CRU (Cocoa Research Unit, Univ. West
Indies, Trinidad) in conjunction with The Maya Mountain
Archaeological Project (MMAP – Cleveland State Univ.)
and is now grown in the international collection of CRU.
- GU255V is a genotype originated from French Guyana,
resistant to Phytophthora palmivora. Inoculation was done
according to Tahi et al. [54]
- PNG seedlings are from a progeny produced in Papua
New Guinea from the cross of two hybrids: 17/3-1 × 36/
3-1, and segregating for Phytophthora resistance. Inocula-
tion was done according to Tahi et al. [54]
- UF676 is a Trinitario genotype tolerant to mirids. Insect
attack was done using protocol described by Babin et al.
[55].
- P7, IMC47, UPA134 are Forastero genotypes originated
from the Upper Amazonian region of Peru, known for
their resistance to Phytophthora palmivora or P. megakarya.
Inoculation was done according to Tahi et al. [54]
- UF 273 is a Trinitario genotype resistant to Monilioph-
thora rorer. Inoculation was done according to Khun et al.
[56]
- 33–49 and BE240 are Nacional genotypes from Ecuador
known for their aromatic and floral taste.
Table 3: Distribution of dimers and trimers motifs in SSRs dataset
Group MotifNumber of SSRs Frenquency
AC
AG
AT
CG
AC/CA/GT/TG
AG/CT/GA/TC
AT/TA
CG/GC
552.4
33.5
14.3
754
323
0
AAT
AAG
AAC
ATG
AGT
AGG
AGC
ACG
ACC
GGC
AAT/ATA/TAA/ATT/TTA/TAT
AAG/AGA/GAA/CTT/TTC/TCT
AAC/ACA/CAA/GTT/TTG/TGT
ATG/TGA/GAT/CAT/ATC/TCA
AGT/GTA/TAG/ACT/CTA/TAC
AGG/GGA/GAG/CCT/CTC/TCC
AGC/GCA/CAG/GCT/CTG/TGC
ACG/CGA/GAC/CGT/GTC/TCG
ACC/CCA/CAC/GGT/GTG/TGG
GGC/GCG/CGG/GCC/CCG/CGC
121
308
54
117
5.4
13.7
2.4
5.2
0.4
3.6
3.5
0.4
2.9
0.6
9
81
79
9
66
13

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SSH libraries or direct libraries were constructed from
these genotypes. More information related to these geno-
types is available through the International Cocoa Germ-
plasm Database [57].
Drought Stress Libraries were constructed from total RNA
isolated from leaves and roots of Scavina 6 plants that
were initially grown under standard conditions in a green-
house [58]. Rooted cuttings were generated and grown to
about 6 months old, then were moved into a Conviron
growth chamber and were not watered until leaves were
visibly wilted (approx 36 hours) at which time tissues
were flash frozen in liquid nitrogen.
RNA Extraction
Plant tissues were frozen in liquid nitrogen or placed in
RNA stabilization reagent (RNA later™, Qiagen) and
stored at -20°C before RNA extraction. Approximately
100 mg of plant tissues were crushed in liquid nitrogen
with poly-vinyl-poly pyrrolidone. The powder was trans-
ferred in a tube containing 1 ml of extraction buffer "
TE3D " (14.8 g EDTA, 84.4 g Tris, 20 g Nonidet P-40, 30 g
lithium dodecyl sulfate, 20 g sodium deoxycholate, 95 ml
H2O) [59]. After 15 min incubation at room temperature,
1 ml of sodium acetate (3 M) and one volume of chloro-
formisoamyl alcohol (24:1) were added. Purification of
the aqueous phase was carried out following centrifuga-
tion by adding one volume of mixed alkyl tri-ethyl ammo-
nium bromine solution (2% MATAB, 3 M NaCl) followed
by 15 min at 74°C. The residual polysaccharides were
then eliminated by addition of one volume of chlorofor-
misoamyl alcohol (24:1) and centrifugation; the aqueous
phase was precipitated by the addition of one volume of
isopropyl alcohol. After centrifugation, the pellet was
resuspended in 50 μl of ribonuclease free water contain-
ing 1 μl of ribonuclease inhibitor (RiboLock™, Fermen-
tas).
RNA samples from cacao tissues were isolated following
the procedure of Charbit et al [59] with modifications.
Following DNase treatment (DNase I, Fermentas), RNA
was then extracted with the phenolchloroformisoamyl
alcohol (25:24:1) step and precipitated with one-tenth
volume of 3 M sodium acetate, pH 5.3, and 2.5 volumes
of 100% ethyl alcohol. An aliquot of RNA was then run by
elecrophoresis on a 1.2% agarose gel and stained with
ethidium bromide to confirm RNA integrity.
Construction of full-length enriched cDNA library
First strand cDNA were synthesized using the Clontech
BD SMART PCR cDNA Synthesis KIT (cat No 634902) as
recommended by the supplier. 0.5–1 μg of total RNA was
incubated at 72°C for 2 min with 1 μl 3' BD SMART CDS
Primer II A (12 μM) and 1 μl BD SMART II A Oligonucle-
otide (12 μM) in a total volume of 5 μl. Then 2 μl 5× First-
Strand Buffer, 1 μl DTT (20 mM); 1 μl dNTP Mix (10 mM
of each dNTP), 1 μl BD PowerScript Reverse Transcriptase
were added and the mix was incubated at 42°C for 1 hour
in an air incubator. According to Glen K Fu (2003) [60], 3
μl Biotin-dATP (Invitrogen), 3 μl Biotin-dCTP (Invitro-
gen), 1 μl 5'-NVVVVV-3' primer 30 μM (50 ng), 2 μl 5×
First-Strand Buffer, 1 μl BD PowerScript Reverse Tran-
scriptase were added, and the mix was kept at 42°C for 30
min. For capture of the unfinished strand, the reaction
was mixed with 600 μl of Streptavidine MagneSphere Par-
amagnetic Particles (Promega) and eluted as recom-
mended by the supplier.
A 2 μl aliquot from the first strand synthesis was used for
the cDNA Amplification by LD PCR (Clontech). Each
reaction was performed with 80 μl deionized water, 10 μl
10× BD Advantage 2 PCR Buffer, 2 μl 50× dNTP Mix (10
mM of each dNTP), 4 μl 5' PCR Primer II A (12 μM), 2 μl
50× BD Advantage 2 Polymerase Mix in a 98 μl total vol-
ume. The PCR reaction consisted of 18 to 25 PCR cycles at
95°C for 15 sec, 65°C for 30 sec, 68°C for 6 min, follow-
ing with a final extension at 70°c for 10 min.
After comparison of fragment sizes with those of model
species (rice and Arabidopsis), fragment sizes of some
cDNA libraries were improved using cDNA size fractiona-
tion. These libraries were submitted to an "agarase step"
[61] after 18 cycles PCR. Double-stranded cDNA was sep-
arated on 1% low-melting agarose gel and the DNA ladder
"lane" was stained and photographed with a ruler. Two
size fractions (< 1.2 kb and > 1.2 kb) were excised from
the unstained cDNA "lane" based on the DNA ladder
"lane". cDNAs were extracted from the gel slices with agar-
ase (Fermentas) according to the supplier instructions.
After a gelase digestion, the cDNA was precipitated with
one volume of isopropanol. The pellets were dried and
suspended in ribonuclease free water. Four to five addi-
tional PCR cycles were performed in order to improve the
efficiency of ligation in pGEM®-T Easy Vector.
For SSH cDNA libraries: The procedure was performed
with the PCR-Select cDNA Substraction kit (Clontech)
according to the manufacturer's recommendations with
slight modifications. The cDNA generated from the
SMART procedure was restricted with 15 U of RsaI (Fer-
mentas) and the two aliquots of the tester cDNA were
ligated to adaptors 1 and 2R, respectively, with 30 U of T4
DNA ligase (Fermentas). The PCR mixture enriched for
differentially expressed sequences was cloned using
pGEMT (Promega) as mentioned above.
One μl of the second strand product was cloned in
pGEM®-T Easy Vector Systems (Promega) and trans-
formed by electroporation in the DH10B T1 resistant
strain of Escherichia coli (Invitrogen); transformation