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ESTs and candidate gene approaches in the Compositae Genome
Richard Michelmore1, Alex Kozik1, María José Truco1, Marta Matviencho2, Oswaldo
Ochoa1, Mireille van Damme1, Dean Lavelle1, Hong Lin2, Barnaly Pande1, Leah
McHale1, Padma Sudarshana1, Jason Argyris1, Paula Ellison1, Kent Bradford1, Louise
Jackson1 and Rick Kesseli3
1Department of Vegetable Crops, University of California, Davis, CA 95616.
2Celera Agen, 1756 Picasso Avenue, Davis, CA 95616.
3Biology Department, University of Massachusetts, Boston MA. 02125.
The Compositae Genome Project (CGP) is in its third year. The initial phase has focused on
sequencing of expressed sequence tag (ESTs). Over 19,000 unigenes of lettuce have been identified, probably
representing at least a third of all genes expressed in lettuce. The current focus is on mapping candidate genes to
agriculturally important phenotypes.
Keywords: Lactuca sativa
, genomics, expressed sequence tag, synteny, candidate gene.
The Compositae Genome Project (CGP) is a collaboration between the laboratories of
Richard Michelmore, Kent Bradford and Louise Jackson (all at the University of California,
Davis), Steven Knapp (Oregon State University, Corvallis), Loren Rieseberg (Indiana
University, Bloomington), and Rick Kesseli (University of Massachusetts, Boston). The
different responsibilities are shown in Figure 1. The CGP builds on long-standing interactions
among these labs and has been running formally since 1999.
Figure 1. Areas of interest and responsibility in the Compositae Genome Project.
• To develop comprehensive gene catalogs for economically important genera of the
Compositae, particularly lettuce and sunflower.
CGN 2003. Eucarpia Leafy Vegetables 2003
(eds. Th.J.L. van Hintum, A. Lebeda, D. Pink, J.W. Schut)
• To develop detailed genetic maps integrating phenotypic data for agriculturally important
traits with candidate gene sequences.
• Determine the extent of synteny between lettuce and sunflower and to Arabidopsis
well as to other plant species such as tomato.
• To enhance the introgression of agriculturally useful alleles from wild species.
• To establish tools and resources for lettuce and sunflower that will be the basis of
genomic investigations in these genera and in the Compositae more generally.
• To understand genome evolution and phenotypic diversification in the Compositae.
The article reviews the progress with respect to lettuce. Parallel progress has been made with
sunflower but is not the subject of the current paper. More details can be found at
The Compositae (Asteraceae) is one of the largest and most diverse families of flowering
plants, comprising one-tenth of all known Angiosperm species. It is characterized by the
compound inflorescence that has the appearance of a single "composite" flower. The
Compositae is divided into two major subfamilies and one minor subfamily with 1,100 to
2,000 genera and over 20,000 species (Cronquist, 1977; Jansen et al.,
1991). The family has
undergone extensive diversification producing a cosmopolitan array of taxa. Compositae are
found in diverse habitats; anaerophytic, xerophytic, and halophytic specialists thrive in some
of the more inhospitable habitats (vertisols, deserts, and salt marshes). The size and adaptive
success of the Compositae have stimulated considerable research into its systematics and
evolution. However, molecular characterization has lagged behind other families (Kesseli and
Multiple species have been domesticated within the Compositae including over 40
economically important species (Kesseli and Michelmore, 1997). These include food (lettuce,
chicory, Jerusalem artichoke), oil (sunflower, safflower), medicinal (Echinacea
and many ornamental (chrysanthemum, dahlia, zinnia, marigold) crops. High quality edible
oils are low in saturated and high in mono- and di-unsaturated fatty acids. The Compositae are
renowned for their variety of novel secondary chemicals including several novel industrial
fatty acids (Caligari & Hind, 1996). The family is also a rich source of powerful insecticides
and industrial chemicals, e.g., pyrethrum (Chrysanthemum
) and rubber (guayule) (Heywood
., 1977). Despite this interesting diversity, individual members of the Compositae have
not been extensively studied genetically and until recently there had been minimal investment
in developing genomics resources. Lettuce and sunflower are representatives of each of the
two major subfamilies and are the best genetically characterized members of the Compositae. Genetic Analysis
In collaboration with others, we have developed several genetic maps (Landry et al
Kesseli et al
., 1994; unpublished data). One inter-specific cross L. sativa
cv. Salinas x L.
UC96US23 is now the core mapping population for lettuce. In collaboration with
Keygene and others, this population has so far been analyzed for 750 AFLP markers, 80 SSR
loci, 51 ESTs (see below) as well as markers for the disease resistance clusters. We have
completed the development of 115 F8 recombinant inbred lines (RILs) from this population
and an additional 185 families are at F6. We now have an integrated map of over 1,400
markers and 9 linkage groups (R. Michelmore et al
., unpublished). The core mapping
population has been adopted by the European ANGEL Project (www.plant.wageningen-
ur.nl/projects/angel/) and the RILs have been distributed to several groups for additional
mapping of markers and phenotypic traits.
Numerous horticultural and morphological traits have been and are being analyzed
genetically. We have conducted quantitative trait locus (QTL) analyses of many traits
including bolting, root architecture, and the ability to extract water from different levels of the
soil profile (Johnson et al
., 2000 & unpublished). We identified several major loci for bolting,
including one with an allele for slow bolting from the wild parent as well as a potential QTL
for tipburn sensitivity. We have identified and mapped many genes for resistance to several
diseases (e.g. Kesseli et al
., 1993, 1994; Maisonneuve et al
., 1994; Robbins et al.
We are characterizing several genes of horticultural importance at the molecular level.
Our emphasis has been on disease resistance genes, particularly the major cluster that contains
and confers resistance to downy mildew. This has involved combinations of map-based
cloning, mutagenesis, and candidate gene approaches (Meyers et al
., 1998a, b; Shen et al
1998, 2002). Dm3
homologs comprise a large family of NBS-LRR (nucleotide binding site-
leucine rich repeat) encoding genes. As part of the Dm3
cloning strategy, we utilized PCR
with degenerate oligonucleotide primers designed from resistance genes cloned from other
species (Shen et al
., 1998 & unpublished). At least 22 distinct families of RGC sequences
have so far been identified. Several families had greater similarity to resistance genes from
other species than to the other lettuce RGC sequences indicating that the different RGC
families originated early in angiosperm evolution. Two families mapped to clusters of known
resistance genes. The Current CGP
Establishment of an extensive EST database for lettuce.
EST libraries were made using a modified SMARTTM (Clontech) approach. cDNAs were
made from ten pools of RNA from different tissues/developmental stages/environmental
conditions of each of L. sativa
cv. Salinas x L. serriola
UC96US23, the two genotypes that
had been used as parents for the core mapping population. For each genotype, each cDNA
was made using oligonucleotide primers that incorporated unique 5’ and 3’ sequence tags so
that the source of each sequence could be subsequently identified. The cDNAs were pooled
and then size-fractionated into four size classes. Each size class was directionally cloned into
a medium-copy vector and transformed separately to reduce size bias.
Table 1. Summary of ESTs developed for lettuce.
Over 68,000 ESTs were generated from L. sativa
cv. Salinas and L. serriola
Over 68,000 lettuce ESTs from the multiple libraries were assembled using CAP3 (Huang &
Madan, 1999) into ~19,000 lettuce unigenes and organized in the CGP mySQL database.
Custom PHP and Python scripts were developed to manipulate the data and view the
assemblies. Our utilization of diverse sources of mRNA and size fractionation strategy
resulted in a very efficient gene discovery (Table 1). This number of unigenes probably
represents at least a third of all genes expressed in lettuce.
Comprehensive EST data are displayed at the CGP web site. This includes the raw
chromatograms, details on BLAST searches, SNP and indel polymorphisms, etc. In addition
to releasing the sequences to GenBank, they were incorporated by TIGR into their latest gene
indices (www.tigr.org/tdb/tgi/) and by MIPS into their SPUTNIK EST database
Development of high-density genetic maps based on transcribed sequences.
We are now focused on mapping numerous ESTs with an emphasis on candidate genes for
agricultural traits (Table 2). Sequences derived from each parent were compared using Python
custom scripts to identify putative indels and single nucleotide polymorphism markers
(SNPs). We have so far identified ~150 indels, 14,000 SSRs and 1,500 SNPs for lettuce. Wet
lab experiments have confirmed the predicted indel polymorphisms in ~90% cases and SNP
polymorphisms in ~70% cases. So far 51 ESTs have been mapped in lettuce using several
technologies. Indel polymorphisms have been analyzed using agarose and acrylamide
electrophoresis. Lettuce SNPs are being analyzed using temperature gradient capillary
electrophoresis (TGCE; REVEALTM, http://www.spectrumedix.com/Reveal.htm).
In order to maximize mapping efficiency, we utilized MapPop (Vision et al
., 2000) and
our software Genoplayer (http://compgenomics.ucdavis.edu/genoplayer.htm) to identify a
subset of the most informative individuals so that marker and phenotypic analyses have only
to be run on 46 individuals with little loss of genetic information. We currently have a
pipeline to map ~ 20 ESTs/week including SNP discovery and validation.
Table 2. Summary of Candidate Clones for Traits in Lettuce.
a putative from database or experimentally confirmed, analysis on going.
Studies of synteny between lettuce and sunflower and to Arabidopsis.
This phase of the project has only been initiated recently, as it required the availability of the
EST sequences. To study macrosynteny, we have identified a COS (conserved ortholog
sequence) set using a new clustering program, Graph9, and visualized using GenomePixelizer
(Kozik et al., 2002; www.atgc.org/GenomePixelizer). About 1,200 lettuce putative COS
(cgpdb.ucdavis.edu/database/est_vs_ath/arabidopsis_cos_map.html); subsets of these are
currently being mapped. In addition, we are also utilizing the candidate genes as part of the
We have also sequenced the majority of our 158 previously mapped RFLP markers. Of
these, ~80% had significant sequence similarity to orthologous or paralogous loci in
To study microsynteny, lettuce and sunflower EST assemblies were BLASTed against
genome then the hits displayed as a linear graphical representation along the
genome. Each element represents a predicted ORF ordered according to position
on the chromosome and there are links to each EST in the CGP database
(cgpdb.ucdavis.edu/database/est_vs_ath/tigr_vs_let_and_sun.html). This has provided further
candidate clusters of sequences that we are now mapping. Databases
We have developed a series of public databases to facilitate ready access to the data. Lettcv
(compositdb.ucdavis.edu/database/lettcv2/display) contains information on over 4,500
cultivars. The Compositae Genome Project site (compgenomics.ucdavis.edu) provides access
to extensive EST information. Compositdb (compositdb.ucdavis.edu) contains the genetic
map information and images for markers as well as descriptions of RFLP probes and PCR-
based markers. Future Directions of the CGP
Subject to funding, the next phase of the CGP will have the following objectives:
Extension of the EST database.
This will involve sequencing more ESTs from four species each of Lactuca
well as single genotypes of safflower and chicory with the primary goal of comprehensive
gene discovery and identification of allelic diversity.
Expansion of high-density genetic maps based on transcribed sequences.
Over 2000 ESTs will be added to the consensus maps. This will establish genetic correlations
Analyses of synteny between lettuce, sunflower, tomato and Arabidopsis.
We will continue the analysis of macrosynteny and microsynteny based on genetic analysis as
well as sequencing of BACs from syntenic regions in lettuce and sunflower.
Phenotypic and molecular analysis of natural variation in six genera.
This will examine variation for a spectrum of morphological and physiological traits at the
phenotypic level and assess natural sequence variation for ~100 genes in 182 accessions
distributed across the five and seven species of Lactuca
respectively, as well
as four wild and cultivated representatives of safflower, chicory, Echinacea and
chrysanthemum. This will establish patterns of selection and indicate potential correlations
between sequence and phenotype as well as the relative contributions of recombination and
We thank the USDA Initiative for Future Agricultural Food Systems program for financial
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[R1]The same situation with Leah. See next comment[R2]I understood Padma did these and she was left out in the poster. I see Barnaly in the co-
author list, I think you need to revise the list.
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Table 3. County vouchers needed at WTU herbarium, arranged alphabetically by county Adams County (42 taxa) Adonis aestivalis Monitor Aegilops cylindrica C Cardaria draba C Carduus nutans B Cenchrus longispinus B Centaurea solstitialis B Centaurea diffusa B Centaurea calcitrapa A Centaurea stoebe B Centaurea repens B Chondrilla juncea B Cirsium arvense C Cirsium vulgare C Conium