# The tomato sequencing project, the first cornerstone of the International Solanaceae Project (SOL)

L.A. Mueller, S.D. Tanksley, J.J. Giovannoni, J. van Eck, S. Stack, - Doil
Choi, - Byung Dong Kim, - Mingsheng Chen, - Zhukuan Cheng, - Chuanyou
Li, et al.

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## To cite this version:

L.A. Mueller, S.D. Tanksley, J.J. Giovannoni, J. van Eck, S. Stack, et al.. The tomato sequencing project, the first cornerstone of the International Solanaceae Project (SOL). Comparative and Functional Genomics, 2005, 6 (3), pp.153-158. 10.1002/cfg.468 . hal-02680359

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Conference Paper

# The Tomato Sequencing Project, the first cornerstone of the International Solanaceae Project (SOL)

†Lukas A. Mueller¹*, Steven D. Tanksley¹, Jim J. Giovannoni², Joyce van Eck², Stephen Stack³, Doil Choi⁴, Byung Dong Kim⁵, Mingsheng Chen⁶, Zhukuan Cheng⁶, Chuanyou Li⁶, Hongqing Ling⁶, Yongbiao Xue⁶, Graham Seymour⁷, Gerard Bishop⁸, Glenn Bryan⁹, Rameshwar Sharma¹⁰, Jiten Khurana¹¹, Akhilesh Tyagi¹¹, Debasis Chattopadhyay¹², Nagendra K. Singh¹³, Willem Stiekema¹⁴, P. Lindhout¹⁵, Taco Jesse¹⁶, Rene Klein Lankhorst¹⁷, Mondher Bouzayen¹⁸, Daisuke Shibata¹⁹, Satoshi Tabata¹⁹, Antonio Granell²⁰, Miguel A. Botella²¹, Giovanni Giuliano²², Luigi Frusciante²³, Mathilde Causse²⁴ and Dani Zamir²⁵

†A full list of affiliations appears at the end of this paper

## Abstract

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the ‘International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation’. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae — and beyond — achieves a context that will facilitate a systems biology approach. Copyright © 2005 John Wiley & Sons, Ltd.

<footnote>*Correspondence to:
Lukas A. Mueller, Department of
Plant Breeding, Cornell University,
Ithaca, NY, USA.
E-mail: Lam87@cornell.edu</footnote>
## Introduction

The Solanaceae, also called nightshades, are a large family of more than 3000 species, including the tuber-bearing potato, a number of fruit-bearing vegetables (tomato, eggplant, peppers), ornamental plants (petunias, *Nicotiana*), plants with edible leaves (*Solanum aethiopicum*, *S. macrocarpon*) and medicinal plants (e.g. *Datura*, *Capsicum*) [1]. The Solanaceae are the third most economically important plant taxon, and the most valuable in terms of vegetable crops. They are also the most variable of crop families in terms of agricultural utility. The closely related coffee (Rubiaceae) is a highly valuable commodity worldwide. In addition to their role as important food sources, many solanaceous species play a role as model plants, such as tomato and pepper for the study of fruit development [2–9], potato for tuber development [10,11], petunia for the analysis of flavonoids, and tomato and tobacco for plant defence [12–16]. The nightshades have also attracted interest because they produce a number of secondary metabolites, some of which have medicinal properties. The Solanaceae are remarkable in that the gene content of the different species is similar despite the markedly different phenotypic outcomes, making the Solanaceae an excellent model for the study of adaptation to natural and agricultural environments [17]. Many Solanaceae share a basic set of 12 chromosomes and are diploid, indicating an absence of large genome duplications and polyploidizations during the evolutionary history of this family. These intrinsic features of the Solanaceae and closely related species such as coffee make them well suited to address fundamental questions in plant biology.

In November 2003, the International Solanaceae Project (SOL) was initiated at a meeting near Washington, where goals were established for Solanaceae research for the next decade ([http://sgn.cornell.edu/solanaceae-project/](http://sgn.cornell.edu/solanaceae-project/)). The ultimate aim of the SOL project is to address two of the most significant questions in plant biology and agriculture: (a) how can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet?; and (b) how can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? To answer these questions in the context of the Solanaceae,

it is necessary to link traits to sequence, requiring both extensive phenotyping [18] and sequence information. It would be desirable to obtain as many full solanaceous genome sequences as possible for direct comparison. Due to the high cost of sequencing complete genomes at high quality, this is not feasible at present. The alternative is to fully sequence a high quality reference genome, and to map onto it ‘cheaper’ sequence data, such as ESTs, methyl-filtered sequences [19–21] or low Cot sequences [22,23] from other species. The availability of good comparative maps [24–26] between many solanaceous plants and the large numbers of EST sequences already available [27] is a great benefit in this approach. Thus, sequencing the gene-rich regions of the tomato genome will be the first cornerstone of the SOL project. After sequencing two rosids, *Arabidopsis* [28] and *Medicago* [29], sequencing a solanaceous plant will shed light on a genome of the more distantly related asterid clade, which will permit comparisons between genomes at longer evolutionary distances and thereby help define a larger view of plant evolution. All the information generated under the SOL umbrella will be made available in a comprehensive website, where all information will be interlinked such that, ultimately, the comparative biology of the Solanaceae will become available in a context that will facilitate a systems biology approach to understanding genome evolution, plant development and plant responses to the environment.

## Tomato genome structure and sequencing method

Approximately three-quarters (730 Mb) of the 950 Mb tomato genome exists as pericentromeric heterochromatin. The remaining one-quarter (220 Mb) of the tomato genome consists of the distal, euchromatic segments of the chromosomes. The DNA found in heterochromatin is rich in repetitive sequences and poor in genes, making it difficult to sequence. The euchromatin is thought to contain mostly single copy sequences and includes more than 90% of the genes, making it relatively easy to sequence. Therefore, the strategy is to sequence only the euchromatic portion of the genome to cover most of the gene space. Sequencing 220 Mb of the tomato genome is therefore a little less
than twice the effort of sequencing the *Arabidopsis*
genome at 150 Mb [30].

The ordered BAC approach was chosen for
sequencing the tomato genome. All sequencing will
be done using standard BAC libraries: the well-
characterized *Hind*III library [31], plus two addi-
tional libraries that the US part of the sequencing
project will provide; all libraries are or will be
deep BAC end-sequenced by the US project, for a
total of 400 000 BAC end sequences. All sequence
will be derived from *Solanum lycopersicum* var
Heinz 1706, as this was the basis for the origi-
nal *Hind*III library. Sequencing will be based on
the F2-2000 map (http://sgn.cornell.edu/), which
has been used as the basis for anchoring 1500
markers by overgo (overlapping oligo) hybridiza-
tion [32]. Currently, more than 650 unambiguous
anchor points are available, but further analysis will
increase this number to an estimated 800–1000
anchor points. In addition, the F2-2000 map is
being combined with an AFLP map, containing
more than 1200 markers, generated at Keygene
in The Netherlands, providing even more anchor
points. For each anchor point, one of the many
anchored BACs will be selected as a ‘seed’ and
sequenced. The tiling path will then be generated
by walking out from this seed in both directions,
using the deep BAC end-sequencing data available
for the BAC libraries. Information from the finger-
print contig (FPC) map available for the *Hind*III
library will also be used. FISH analysis will be used
to confirm chromosome mappings and delineate
the euchromatin/heterochromatin boundaries [33];
anchored BACs will also be mapped on IL lines
to verify location. Other methods available for full
genome sequencing have also been assessed. Full
genome shotgun sequencing is not a cost-effective
way to sequence a fraction of the genome. Methyl
and Cot filtering are both methods providing a
bias for coding sequence and therefore euchromatic
sequence. All these methods do not by themselves
provide gene order, which will be critical for a ref-
erence genome.

Currently, 10 countries are involved in sequenc-
ing the tomato genome. The 12 chromosomes have
been split up between the countries as follows (see
Figure 1): Korea (chromosome 2), China (3), UK
(4), India (5), The Netherlands (6), France (7),
Japan (8), Spain (9), Italy (12) and USA (1, 10, 11).
The US project will also generate the additional
BAC libraries, perform BAC end-sequencing, and

build a data repository as a resource for the entire
project. Argentina will sequence the mitochron-
drial genome, and Italy the chloroplast genome.
The organellar genome sequences will be important
for distinguishing genomic insertions of organellar
sequences from organellar contamination contained
in the BAC libraries.

Bioinformatics

In such a large project, bioinformatics plays a
crucial role. In particular, the sequence quality
standards and the annotation have to be uni-
form across the different chromosomes generated
through different national projects, and efficient
data access has to be provided to the scientific
community. A SOL bioinformatics committee com-
prised of participating sequencing country repre-
sentatives is working on a bioinformatics guide-
line document that should be followed by all
project members to ensure that results are com-
parable in the entire genome. The guidelines are
available from http://sgn.cornell.edu/solanaceae-
project/. In addition, the following important con-
ventions have been adopted: (a) the original data
has to be stored for all data types, such as
all chromatograms and assembly files from the
BAC sequencing, all supporting data for gene
calls etc; (b) all data has to be traceable to the
submitters and data generators; and (c) a uni-
fied web resource will be created that makes
all data accessible to the users without delay
and in an intuitive format (‘one-stop-shop’). Sev-
eral centres will work together to implement the
portal, including MIPS (http://mips.gsf.de/), VIB
Ghent, Wageningen University and the Research
Centre, KRIBB (http://www.kribb.re.kr), Kazusa
(http://www.kazusa.or.jp/), SGN and centres in
other countries. All sequence data will be submitted
to GenBank, and to the Solanaceae Genomics Net-
work (SGN; http://sgn.cornell.edu/), which will
serve as a repository and access point for the
data.

Conclusions

Sequencing the tomato genome offers exciting
new perspectives and opportunities for plant biol-
ogy. The sequence will be compared to other
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Overview of the International Tomato Sequencing Project. The 12 tomato chromosomes are sequenced by an international consortium of 10 countries [Korea (chromosome 2), China (3), UK (4), India (5), The Netherlands (6), France (7), Japan (8), Spain (9), Italy (12) and USA (1, 10, 11)], as shown. This overview is available from the SGN website ([http://sgn.cornell.edu/help/about/tomato_sequencing.html](http://sgn.cornell.edu/help/about/tomato_sequencing.html)) and will be continuously updated as sequencing progresses. More information on the project is available on the page</figcaption></figure>

sequenced genomes such as *Arabidopsis* and rice to inform us of the evolutionary history of these plants. In conjunction with sequence data for other solanaceous plants, the tomato sequence will be the basis for investigating the phenotypic diversity and comparative biology in the Solanaceae, one of the main aims of the SOL project. This will shed light on mechanisms of gene regulation, evolution, signalling, disease resistance and defence, and fruit development and quality, and finally contribute to the improvement of agriculture through the phenotypic diversity found in nature.

## Full List of Author Affiliations

1. Department of Plant Breeding, Cornell University, Ithaca, NY, USA

2. Boyce Thompson Institute, Ithaca, NY, USA

3. Department of Biology, Colorado University, Fort Collins, CO, USA

4. KRIBB, Taejon, Korea

5. Seoul National University, Seoul, Korea

6. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

7. Warwick HRI, Wellesbourne, UK
8. Imperial College, London, UK

9. SCRI Invergowrie, Dundee, UK

10. University of Hyderabad, Hyderabad, India

11. University of Delhi South Campus, New Delhi, India

12. National Centre For Plant Genome Research, New Delhi, India

13. NRC on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India

14. Centre for BioSystems Genomics, Wageningen, The Netherlands

15. University of Wageningen, Wageningen, The Netherlands

16. Keygene, NV., Wageningen, The Netherlands

17. Plant Research International, Wageningen, The Netherlands

18. Institut National Polytechnique de Toulouse, Toulouse, France

19. Kazusa, Kisarazu, Chiba, Japan

20. Instituto de Biologia Molecular y Celular de Plantas Valencia, Spain

21. University of Malaga, Malaga, Spain

22. ENEA, Rome, Italy

23. U Naples, Naples, Italy

24. INRA, Avignon, France

25. Hebrew University, Jerusalem, Israel

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