# ASolanum lycopersicum×Solanum pimpinellifolium Linkage Map of Tomato Displaying

Authors: Arun Sharma; Liping Zhang; David Niño-Liu; Hamid Ashrafi; Majid R. Foolad
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

We have identified an accession (LA2093) within the tomato wild species Solanum pimpinellifolium with many desirable characteristics, including biotic and abiotic stress tolerance and good fruit quality. To utilize the full genetic potential of LA2093 in tomato breeding, we have developed a linkage map based on anF2  population of a cross between LA2093 and a tomato breeding line, using 115 RFLP, 94 EST, and 41 RGA markers. The map spanned 1002.4 cM  of the 12 tomato chromosomes with an average marker distance of 4.0 cM. The length of the map and linear order of the markers were in good agreement with the published maps of tomato. The ESTs were chosen based on their sequence similarities with known resistance or defense-response genes, signal-transduction factors, transcriptional regulators, and genes encoding pathogenesis-related proteins. Locations of several ESTs and RGAs coincided with locations of several known tomato resistance genes and quantitative resistance loci (QRLs), suggesting that candidate-gene approach may be effective in identifying and mapping new R genes. This map will be useful for marker-assisted exploitation of desirable traits in LA2093 and other S. pimpinellifolium accessions, and possibly for utilization of genetic variation within S. lycopersicum.


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## Body

## 1. Introduction

The
tomatoSolanum species (Solanum subsection Lycopersicon) include the cultivated tomato, S. lycopersicum L. (formerly Lycopersicon
esculentum Miller), and more than 10 related wild species (http://www.sgn.cornell.edu/about/solanum_nomenclature.pl). It is
estimated that S. lycopersicum accounts for only about 5% of the total genetic variability in the tomato gene
pool [1]. Conversely, the tomato wild
species bear a wealth of genetic variability for many agriculturally and
biologically important characteristics. During the past several decades, tomato
wild species have been utilized extensively in traditional breeding programs,
however, mainly for improvement of simply inherited traits such as vertical
disease resistance. Genetic variation in the wild species for complex traits
such as tolerance to environmental stresses, quantitative disease resistance,
and fruit yield and quality has remained largely unexploited [2]. This is mainly due to the
inadequacy of traditional breeding protocols to identify, select, and
successfully transfer genes controlling such complex traits. The identification
of genes underlying quantitative characters is often difficult, particularly if
their phenotypic effects are unrecognizable from the phenotype [3]. Furthermore, transfer of
desirable genes from wild species into elite breeding lines is not without
inherent difficulties. Upon interspecific hybridization, a major task becomes  eliminating
the great bulk of undesirable exotic genes while maintaining and selecting for
desirable characteristics. These limitations, however, may no longer be
insurmountable with the advent of molecular biology tools such as genetic
markers and maps and marker-assisted selection (MAS). Among various advantages,
molecular markers and maps can facilitate determination of the number,
chromosomal location, and individual and interactive effects of genes (or
quantitative trait loci (QTL)) that affect complex traits. Following their
identification, desirable genes or QTLs can be introgressed into the cultigen
and undesirable characteristics eliminated by foreground and background MAS.During the past few decades, several molecular linkage maps
of tomato have been developed mainly based on interspecific crosses between the
cultivated and related wild species of tomato (for a complete list see Foolad
2007). The first molecular linkage map of tomato was published in 1986, which
included 18 isozyme
and 94 DNA markers [4]. The high-density linkage map of
tomato, which originally was developed based on an F2 population of
a S. lycopersicum  ×  S. pennellii cross and 1030 molecular markers [5], currently comprises more than
2000 markers with intermarker spacing of ≤1 cM (http://www.sgn.cornell.edu/cview/map.pl?map_id=9). The high
level of molecular marker polymorphism between S. lycopersicum and S.
pennellii facilitated the development of this high-density map. With this
genetic map, it is likely that any gene of interest would be within one to a
few centiMorgan (cM).
However, many important agricultural traits are not segregating in this
population and many of the markers in this map are not polymorphic in other
populations of tomato, in particular those derived from intraspecific crosses
within the cultigen or between the cultigen and closely related wild species
such as S. pimpinellifolium L. (formerly L. pimpinellifolium (L.) Miller) and S. cheesmaniae (L. Riley) Fosberg (formerly L. cheesmaniae L. Riley). For
example, it has been determined that only ~30% of the RFLP markers in the
high-density map detect polymorphism in S.
lycopersicum  ×  S.
pimpinellifolium populations following digestion of genomic DNAs with many
restriction enzymes [6, 7]. In a more recent study, only
less than 15% of the RFLP markers from the high-density map detected
polymorphism between a Mexican accession of S.
pimpinellifolium and a S.
lycopersicum breeding line (MR Foolad et al., unpubl.). Such low levels of
marker polymorphism necessitated the development of several species-specific
molecular maps, as listed elsewhere [2]. Among the different wild
species of tomato, however, genetic maps developed based on crosses between the
cultivated tomato and S. pimpinellifolium would be more useful for practical purposes, as described below.S. pimpinellifolium is the only red-fruited wild species of tomato and the only
species from which natural introgression into the cultivated tomato has been
detected [8]. In addition, during the past
several decades, extensive genetic introgressions from this species into the
cultivated tomato have been made through plant breeding [8–10]. Accessions within S. pimpinellifolium are highly
self-compatible and bidirectionally cross-compatible with the cultivated
tomato. Because of the close phylogenetic relationships between the two
species, there is little or no difficulty in initial crosses or in subsequent
generations of prebreeding and breeding activities. Furthermore, S. pimpinellifolium harbors numerous
desirable horticultural and agronomic characteristics, including disease
resistance [11–13], abiotic stress tolerance [14, 15], and good fruit quality [2, 16], and much fewer undesirable
traits than most other wild species of tomato. However, to utilize the full
genetic potential of this species, it is necessary to detect molecular
polymorphisms between this species and the cultivated tomato. Detection or
development of polymorphic markers, in particular functional markers (see
below), and construction of new molecular linkage maps based on desirable S. lycopersicum  ×  S. pimpinellifolium crosses are a step
toward genetic exploitation of this species. Furthermore, because of extensive
introgressions from S. pimpinellifolium into
modern cultivars of tomato, such
markers and maps will also be useful when exploiting the available genetic
variation within the cultigen.Most of the previous
genetic linkage maps of tomato were constructed based on random genetic markers
such as RFLPs, RAPDs, AFLPs, and SSRs. Recently, however, DNA sequences based
on expressed sequence tags (ESTs) and resistance gene analogs (RGAs) have
become available, which can be used to develop genetic markers and maps or used
as candidates to identify functional genes. Development of markers and maps
based on informative sequences will be useful for identification and
potentially cloning of genes and QTLs of agricultural and biological
significance. ESTs are generally derived from cDNA clones and may have
applications in gene sighting, genome mapping, and identification of coding
regions in genomic sequences. While ESTs can serve the same purposes as random
DNA markers, they provide the additional feature of pointing directly to
expressed genes and thus can expedite gene discovery and comparative genomics.
The growing EST databases in different plant species, including tomato, have
provided valuable resources for development of EST-based markers. The
association of EST markers with phenotypes can lead to a better understanding
of biochemical pathways and mechanisms affecting important traits.
Identification and characterization of RGAs has also been proposed as a candidate-gene
approach to identify genes potentially related to disease resistance [17–21]. Although not all amplified
products may correspond to functional disease resistance genes [21], RGA primers have been shown
to amplify the conserved sequences of leucine-rich repeats (LRR),
nucleotide-binding sites (NBS),
or serine/threonine protein kinases (PtoKin), thereby targeting genes and gene families for disease
resistance, defense response, or other important signal transduction processes [22]. Thus, RGAs have been
considered useful not only as genetic markers but also as potential that leads
to the identification of important genes. During the past decade, RGAs have
been used for mapping of QTLs for many important characters, including disease
resistance.Recently, we identified several accessions ofS. pimpinellifolium (including LA2093)
with desirable horticultural characteristics such as disease resistance,
abiotic stress tolerance, and good fruit quality. To facilitate genetic
characterization and exploitation of LA2093, and possibly other accessions, we
have developed a genetic linkage map based on an F2 population of a
cross between LA2093 and tomato breeding line NCEBR-1 using 250 DNA markers,
including RFLPs, ESTs, and RGAs. Previously, two molecular linkage maps of
tomato based on different crosses between S.
lycopersicum (denoted as L) and S.
pimpinellifolium (denoted as PM) were reported by Grandillo and Tanksley [6] (referred to as L  ×  PM1 map) and Chen and Foolad [7] (referred to as L  ×  PM2 map). The map presented here (referred to
as L  ×  PM3) is different but complementary to the
previous two L  ×  PM maps, as it contains a large number of ESTs
and RGAs along with some new RFLP anchor markers that can facilitate molecular
investigation and exploitation of this and other accessions of S. pimpinellifolium. We have compared
the L  ×  PM3 map with other molecular linkage maps of
tomato and discussed similarities and differences in relation to phylogenetic
relationships between parents of the various mapping populations.


## 2. Materials and Methods

### 2.1. Plant Materials and Mapping Population

Inbred
sources of NCEBR-1 (S. lycopersicum)
and LA2093 (S. pimpinellifolium) were
hybridized and F1 progeny produced. NCEBR-1 (PVP) is a
horticulturally superior, multiple disease resistant, advanced tomato breeding
line received from RG Gardner, University of North Carolina, Fletcher, NC, USA. A single F1 hybrid plant was self-fertilized to produce F2 seed. A total of 900
F2 individuals were grown under field conditions and screened for
various characteristics. Among other traits, the population was segregating for
growth habit (determinate versus indeterminate). Indeterminate growth habit is
an undesirable characteristic with confounding effects on other characteristics
such as disease resistance and fruit quality. To obtain a population suitable
for QTL mapping and breeding purposes, indeterminate plants were eliminated. A
total of 172 F2 individuals, hereafter referred to as the L  ×  PM3 F2 population, were chosen and
grown to maturity and used to construct the molecular linkage map.


### 2.2. RFLP Analysis

Nuclear
DNA was extracted from approximately 10 g of leaf tissue from each of the
parental lines and F2 individuals using standard protocols for
tomato [23, 24]. Genomic DNAs were treated with RNase
and digested with eight restriction enzymes, including DraI, EcoRI, EcoRV, HaeIII, HindIII, RsaI, Sca1, and Xba1 following manufacturers’ instructions, and parental
polymorphism survey blots were prepared. To identify sufficient number of
polymorphic anchor RFLP markers to develop a framework linkage map, parental
survey blots were probed with a total of 340 random
tomato genomic (TG) or cDNA (CD or CT) clones, originally chosen from the
high-density molecular linkage map of tomato [25]. Agarose gel electrophoresis,
Southern blotting, hybridizations, and autoradiography were conducted as
described elsewhere [26]. Probes were labeled with [32P]dCTP
by primer extension [27]. Following identification of
polymorphic RFLP markers (see Section 3 for rates of polymorphism), genomic DNAs of the 172 F2 individuals were digested with the 8 restriction enzymes and multiple sets of
Southern blots were prepared. Blots were hybridized with clones detecting
polymorphism and a total of 115 RFLP markers were scored in the F2 population.


### 2.3. EST Analysis

A set of unique ESTs was selected from the tomato gene index sources
maintained by The Institute for Genomic Research (TIGR;http://www.tigr.org/) (now at the Computational Biology and Functional
Genomics Laboratory at Harvard University; http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/est_report.pl). Each EST represents a valid (partial or complete)
copy of a transcribed functional allele. We selected 140 ESTs from a diverse
array of candidate genes and gene families, many of which are known or assumed
to play roles in disease-resistance or defense-response mechanisms. Among them
we included ESTs with homology to resistance (R) genes, signal transduction genes, transcriptional regulator factors,
and genes encoding pathogenesis-related proteins. We used this targeted
strategy to obtain a set of potentially functional markers for marker-assisted
selection in our tomato-breeding
program. The 140 EST clones, purchased from the
Clemson University Genomics Institute (http://www.genome.clemson.edu/), Clemson, SC, USA, were used as RFLP probes to identify
polymorphism between the two parents. Among them, 96 provided polymorphic
alleles (Table 1). The polymorphic ESTs were
used as RFLP probes to genotype the F2 individuals, examine their
segregation, and map onto the tomato chromosomes.Table 1
Listof ESTs mapped in theSolanum lycopersicum  ×  S. pimpinellifolium F2 population, their putative function,
chromosomal location, and copy
number.
EST cloneSaGN-TDPbutative functionChr.Ccopy no.cTOF3A14C146883Cytosolic Cu, Zn Superoxide dismutase,S. lycopersicum12cTOE7J7aC139397Endo-1,4-beta-glucanase,S. lycopersicum16cLED27E12C19568Cold
acclimation protein WCOR413-like protein form,O. sativa12cTOE6F10C139034Lipoxygenase,S. lycopersicum15cLEG9N2C45935Subunit A
of ferredoxin-thioredoxin reductase,S.
tuberosum11cLES9N20C79709ASC1 (Alternaria stem canker resistance protein),S. lycopersicum11cLEC6O2C11013Polyamine
oxidase,A. thaliana11cTOF20P4C142906Carotenoid
cleavage dioxygenase 1-2,S.
lycopersicum15cLEZ11K12C98684Snakin2
precursor,S. lycopersicum11cTOA9E13C117653Squalene synthase,C.
annuum15cTOA9C11C117644Similar to
WRKY transcription factor Nt-SubD48,N.
tabacum21cLET10E15C79822Acidic 26kDa endochitinase precursor,S. lycopersicum21cTOF19J9C142319Phosphoribosylanthranilate
isomerase,A. thaliana21cLEY1K9C97179Pathogen-inducible
alpha-dioxygenase,N. attenuata24cLEW11E20C89000Resistance complex protein I2C-3,S. lycopersicum27cTOF16A9C141311Calmodulin 3 protein,S. lycopersicum39cLER17H16C71298Elicitor-inducible cytochrome P450,N. tabacum31cTOF18P1C142154Serine palmitoyltransferase,S. tuberosum33cLEX12O16C92852Ethylene response factor 5,S.lycopersicum36cTOE2F15C137984Catalase isozyme 1,S. lycopersicum31cTOF29J22C1454124-coumarate-coA ligase 1,S. tuberosum32cLEX10F20C92172Ethylene response factor 1,S. lycopersicum34cTOF14B17C141010Anthocyanin 5-O-glucosyltransferase,S. sogarandinum41cLED15E5C16128Shikimate kinase chloroplast precursor,S. lycopersicum41cLEN13D5C66215Chorismate synthase 1 precursor,S. lycopersicum44cTOS21D12C163577Similar to
heat shock factor,N. tabacum43cTOF10N11C140057Myo-inositol-1-phosphate synthase,S. lycopersicum44/5cLEW24M21C90911TMV
disease resistance protein-like protein,Cicer
arietinum42cLEW22D11bC903524-coumarate:coenzyme A ligase,N. tabacum410cLER5E19C73560Phospholipase
PLDb1,S. lycopersicum51cTOC2J14aC127676Disease resistance gene homolog Mi-copy1,S.lycopersicum59cTOC2J14bC127676Disease resistance gene homolog Mi-copy1,S.lycopersicum59cTOF26E9C144413Prf,S.
pimpinellifolium52cTOE1K1C136851Spermidine synthase,S. lycopersicum54cTOE7J7bC139397Endo-1,4-beta-glucanase,S. lycopersicum56cTOF29B13C145236Metallothionein-like
protein type 2 a,S. lycopersicum52cTOF33C3C146601Serine/threonine
protein kinase Pto,S. lycopersicum510cTOF23J19C143585Heat shock
protein 90,S. lycopersicum54cLEG32E10C34795Lipoxygenase B,S.
lycopersicum66cTOF8F19C148467Ascorbate peroxidase,S. lycopersicum62cLEZ16H16C99197Contains
similarity to disease resistance response protein,Pisum sativum61cLED11A2C15134Mitogen-activated
protein (MAP) kinase 3,C. annuum62cLEW22D11aC903524-coumarate:coenzyme A ligase,N. Tabacum610cLEY21L21C97473Disease
resistance gene homolog Mi-copy1,S. lycopersicum66cLEW22N22C90504Ethylene-responsive
element binding factor 6-N. sylvestris63cTOF34C13C146804Peroxiredoxin
Q-like protein,A. thaliana71cLEN14F9C66474Sucrose-phosphate
synthase,S. lycopersicum71cTOF21F12C142982Dehydroquinate dehydratase/shikimate, NADP
oxidoreductase,S. lycopersicum79cLEN13G22C662461-aminocyclopropane-1-carboxylate oxidase,S. lycopersicum74cLEY22L20C97674Peroxidase precursor,S. lycopersicum73cTOE15M9C136013MYB-related
transcription factor VlMYBB1-1,Vitis
labrusca  ×  V. vinifera76cLEG34O20C35423UDP-glucose:salicylic acid glucosyltransferase,N. tabacum74cLEN14C8C66419PR-related protein, PR P23 (salt-induced protein),S. lycopersicum83cTOF9D16C148734Pathogenesis-related
protein 5-1,S. lycopersicum81cTOF28D12C144993Polyphenol oxidase E, chloroplast precursor,S.
lycopersicum87cLEN10H3C65539Heat shock
factor protein HSF8 (Heat shock transcription factor 8),S. lycopersicum82cLEI16E21C47449Cold-induced glucosyl transferase,S. lycopersicum83cTOF2N15C145786Osmotin-like protein OSML13 precursor (PA13),S. lycopersicum83cTOE23J12C137767Monodehydroascorbate reductase,S. lycopersicum83cLED27C20C19537DNADPH oxidase; gp91-phox homolog,S. lycopersicum81cLER14J12C70373WRKY
transcription factor IId-1 splice variant 2,S. lycopersicum81cTOF2L16C145747Phenylalanine ammonia-lyase (PAL),S. lycopersicum81cTOD3N7C132799Endo-1,4-beta-glucanase,S. lycopersicum82cLEX11E19C92435Putative NADH-ubiquinone oxireductase,A. thaliana91cTOE10J18C134749PR protein sth-2,S. Tuberosum93cLEC13E21C1592P14 (PR-Protein),S. lycopersicum93cLEC6M14C10964PR-protein sth-2,S. Tuberosum95cLER14J6C70387Hexose transporter,S.
lycopersicum91cTOF19O3C142383Hydroxyproline-rich
glycoprotein homolog,A. thaliana93cLEZ6E21bC100278Ubiquitin,S. lycopersicum104/5cLED18G6C17041Similar to
WRKY-like drought-induced protein,Retama
raetam106cTOD4I20C133021Tyrosine aminotransferase,A. thaliana102cLHT11J12C100975Diacylglycerol kinase,S. lycopersicum102cLER4F5C73337Ferredoxin-I chloroplast precursorS. lycopersicum104cTOF30K21C146034Chloroplast ferredoxin I,S. lycopersicum10>10cTOF22M16C143336NADH-ubiquinone oxidoreductase 23 kDa subunit,S. lycopersicum101cLEN14K6C66563Multiresistance protein homolog,A. thaliana103/2cLEX10N16C92314PR protein,S.
lycopersicum10>10cLEC18O1C3034Basic 30kDa endochitinase precursor,S. lycopersicum10>10cTOF31H10C146231Catechol O-methyltransferase,N. tabacum108cLEN9P2C69374Multiresistance
protein homolog,A. thaliana102cLED13I7C15652Resistance complex protein I2C-1,S. lycopersicum117cTOF28I23C145097Resistance
complex protein I2C-5,S.
pimpinellifolium11>10cLEZ6E21aC100278Ubiquitin,S. lycopersicum114/5cTOF29F6bC14533010-hydroxygeraniol oxidoreductase, -S. lycopersicum117cLEC14I18aC1998Resistance complex protein I2C-2,S. lycopersicum11>10cLEC14I18bC1998Resistance complex protein I2C-2,S. lycopersicum11>10cLEM22K17C627089-cis-epoxycarotenoid dioxygenase,S. lycopersicum117cLED23K21C18512Resistance complex protein I2C-5,S. lycopersicum11>10cLES18N16C76694Phosphatidylinositol 4-kinase,S. tuberosum112cTOS21D14C163579WRKY
transcription factor IId-2,S. lycopersicum121cLPT1G11C109877S-adenosyl-l-homocysteine
hydrolase,S. lycopersicum124cLEZ15E8C98979Extensin class I,S. Lycopersicum12>10cLEW25D9C90989Glutamine synthetase,S. lycopersicum123Saolanaceae Genome Network (SGN) can be accessed at http://www.sgn.cornell.edu/.Tbhe putative function of each EST has been derived
from Computational Biology and Functional Genomics Laboratory web site (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/est_report.pl), used to be maintained at The Institute for Genomic
Research (TIGR). (Computational Biology and
Functional Genomics Laboratory).Tche exact or approximate copy number of ESTs in tomato
genome was determined based on the number of hybridized bands on Southern blot
gels and may be varied in different labs. Where there is a “/” sign, the
figures in the left side denote the number of copies in S. lycopersicum parent and those in the right side denote the
number of copies in S. pimpinellifolium parent.


### 2.4. RGA Analysis

#### 2.4.1. Selection of Primers

Ten pairs of
oligonucleotide primers, previously designed based on conserved LRR, NBS, and
PtoKin motifs of several resistance genes, were used (Table2; [28]). Some primers were chosen to be degenerate at the
redundant third position (3′ end) in the codons to cover a range of possible
sequences encoding the motifs, and thus to increase the efficiency of PCR
amplification [19, 29]. Only one pair of primers was
used for each PCR amplification.Table 2
Oligonucleotide primers
designed based on the conserved amino acid sequences within theLRR, NBS, and Pto protein domains encoded by
various R-genes.
GroupPrimersSequences (5′-3′)aDesign basisReferencesLRRCLRR forTTTTCGTGTTCAACGACGLRR domain of the tomatoCf-9 gene conferring resistance to Cladosporium
fulvum[30]CLRR revTAACGTCTATCGACTTCTNLRR forTAGGGCCTCTTGCATCGTLRR domain of the tobaccoN gene conferring resistance to TMVNLRR revTATAAAAAGTGCCGGACTRLRR forCGCAACCACTAGAGTAACLRR domain in theRPS2 gene conferring resistance to Pseudomonas syringae in ArabidopsisRLRR revACACTGGTCCATGAGGTTXLRR forCCGTTGGACAGGAAGGAGLRR domain of the riceXa21 gene conferring resistance to Xanthomonas campestris pv oryzaeXLRR revCCCATAGACCGGACTGTTANo. 2TATAGCGGCCGCIARIGCIARIGGIARNCCConserved P-loop and hydrophobic
NBS regions of theN and RPS2 genes from tobacco and Arabidopsis respectively[29]ANo. 3ATATGCGGCCGCGGIGGIGTIGGIAARACNACNBSS1GGTGGGGTTGGGAAGACAACGHydrophobic domain and P-loop of
conserved NBSs from theN and RPS2 genes from Arabidopsis and the L6 gene from flax conferring resistance to rust[18, 31]S2GGIGGIGTIGGIAAIACIACAS1CAACGCTAGTGGCAATCCAS3IAGIGCIAGIGGIAGICCPtokin1GCATTGGAACAAGGTGAASerine/threonine protein kinase
domain of thePto gene conferring
resistance to the bacterial pathogen Pseudomonas
syringae pv tomato in tomato[30]Ptokin2AGGGGGACCACCACGTAGPtokin3TAGTTCGGACGTTTACATPtoKinPtokin4AGTGTCTTGTAGGGTATCRLK forGAYGTNAARCCIGARAASerine/threonine kinase sequence subdomains of the wheatLr10 gene conferring resistance to Puccinia recondita[20]RLK revTCYGGYGCRATRTANCCNGGITGICCaCode for mixed
bases: D  =  A/G/T; I  =  Inosine;
N  =  A/G/C/T; R  =  A/G;  Y  =  C/T.


#### 2.4.2. PCR Amplification

PCR conditions for amplification of RGAs were
described elsewhere [11]. Briefly, each amplification was performed in a 25-μL
volume consisting of 300 μM
each of dATP, dCTP, dGTP, and dTTP, 5 mM of MgCl2, 1 unit of Taq DNA polymerase, 2.5 μL of 10X buffer (PCR Core system I; Promega,
Madison, Wis, USA), 2 μL
of each primer, and 40 ng of genomic DNA that was used as template. For control
reactions, the template was substituted with sterile, nuclease-free ddH2O.
All PCR mixtures were overlaid with mineral oil and carried out in a Perkin
Elmer DNA Thermal Cycler 480 (Perkin Elmer, Foster City, Calif, USA), programmed
for 4 minutes at 94°C for an initial denaturation, and 36 cycles of
1 minute at 94°C (DNA denaturation), 1 minute at 50°C
(primer annealing), and 1.5 minutes at 72°C (primer extension),
followed by a final 7-minute extension at 72°C.


#### 2.4.3. Gel Electrophoresis and Silver Staining

Denaturing polyacrylamide gel
electrophoresis (PAGE) was used to separate and detect individual RGA bands [30]. Briefly, a denaturing gel (7 M Urea—6% polyacrylamide) was prepared in a
sequencing gel apparatus (420  ×  330  ×  0.4 mm; Fisher
Biotech, Springfield, NJ, USA) using Bind- and Repel-Silane (Promega). After
polymerization, the gel was prerun in 1X Tris-borate-EDTA (TBE) buffer for 30
minutes at 40 W (~1400 V) to reach a gel temperature of 50°C.
Twelve μL
of loading buffer (10 M Urea—0.08% xylene cyanole) were added to each 25 μL
amplified DNA sample and the mixture was denatured at 95°C for 5
minutes and immediately put on ice. After cleaning the gel-loading surface, a
0.4 mm-thick shark comb (Fisher
Biotech, Springfield, NJ, USA) was inserted into the gel. Subsequently,
7 μL
of each PCR-amplified sample were loaded. Each gel accommodated 60 DNA samples
and three DNA size markers (1 Kb, 100 bp, 50 bp). The gel was run at 35 W (~1350 V) for 3.5–4 hours. After
electrophoresis, the gel, fixed to the Bind-Silane surface of one glass plate,
was silver stained following the manufacturer’s protocol (Promega). The gel was
air dried at room temperature overnight and stored in dark for future scoring
and scanning. All amplifications and gel electrophoresis procedures were
repeated at least once.


#### 2.4.4. Identification of Informative RGA Markers

Following gel
electrophoresis and staining, polymorphic and monomorphic bands were observed.
Polymorphic bands were directly scored as dominant markers and used for genetic
mapping. To determine whether monomorphic bands could detect polymorphism if
used as RFLP probes, they were excised from the gel (as described
in [28, 32]), purified with the QIAgene quick
Gel Extraction Kit (QIAGEN, Valencia, Calif, USA), labeled with 32P-dCTP,
and used to hybridize the parental survey blots. Probes which detected
polymorphism between the two parents were then used to hybridize Southern blots
of the F2 population, and scored as either dominant or codominant
markers. Overall, a total of 43 RGA markers were successfully scored and mapped
onto the 12 tomato chromosomes.


#### 2.4.5. Size Determination of RGA Fragments

PAGE polymorphic and monomorphic
fragments were excised from the dried polyacrylamide gel and reamplified, by
using a needle scratching and PCR reamplification method [32]. The reamplified products and DNA size markers (1 Kb, 100 bp, and 50 bp) were run on a 1.0% agarose gel, stained with ethidium
bromide, and photographed to determine the size.


### 2.5. Statistical and Mapping Analyses

Segregation of the 250 DNA markers (115 RFLPs, 94 ESTs, and 41 RGAs) in the F2 population was tested for deviation from the expected Mendelian genotypic
ratios of 1:2:1 (for codominant) or 1:1 (for dominant markers) using chi-square
(χ2)
goodness-of-fit analysis. Multipoint linkage
analysis of the genetic markers in the F2 population was performed
using the MapMaker program v. 3.0 [33] and a genetic linkage map was
constructed. Briefly, the group command was used to assign markers into linkage
groups using a minimum LOD score of 3.0 and a maximum recombination fraction of
0.20. Three-point linkage analysis was performed to determine the maximum
likelihood recombination fraction and the associated LOD score for each
combination of loci. The “order” and “compare” commands
were used to find the best order of loci within each group, followed by using
the “ripple” command to verify the order. Markers were included
within the framework map only if the LOD value for the ripple was greater than
3.0. Once the linear order of markers along each chromosome was determined,
recombination frequencies between markers were estimated with multipoint
linkage analyses. The Kosambi mapping function [34] was used to convert
recombination frequencies to map distances in cM. The distribution of
percentage of the S. lycopersicum genome (L) in the F2 population was estimated using the computer program Qgene [35].


## 2.1. Plant Materials and Mapping Population

Inbred
sources of NCEBR-1 (S. lycopersicum)
and LA2093 (S. pimpinellifolium) were
hybridized and F1 progeny produced. NCEBR-1 (PVP) is a
horticulturally superior, multiple disease resistant, advanced tomato breeding
line received from RG Gardner, University of North Carolina, Fletcher, NC, USA. A single F1 hybrid plant was self-fertilized to produce F2 seed. A total of 900
F2 individuals were grown under field conditions and screened for
various characteristics. Among other traits, the population was segregating for
growth habit (determinate versus indeterminate). Indeterminate growth habit is
an undesirable characteristic with confounding effects on other characteristics
such as disease resistance and fruit quality. To obtain a population suitable
for QTL mapping and breeding purposes, indeterminate plants were eliminated. A
total of 172 F2 individuals, hereafter referred to as the L  ×  PM3 F2 population, were chosen and
grown to maturity and used to construct the molecular linkage map.


## 2.2. RFLP Analysis

Nuclear
DNA was extracted from approximately 10 g of leaf tissue from each of the
parental lines and F2 individuals using standard protocols for
tomato [23, 24]. Genomic DNAs were treated with RNase
and digested with eight restriction enzymes, including DraI, EcoRI, EcoRV, HaeIII, HindIII, RsaI, Sca1, and Xba1 following manufacturers’ instructions, and parental
polymorphism survey blots were prepared. To identify sufficient number of
polymorphic anchor RFLP markers to develop a framework linkage map, parental
survey blots were probed with a total of 340 random
tomato genomic (TG) or cDNA (CD or CT) clones, originally chosen from the
high-density molecular linkage map of tomato [25]. Agarose gel electrophoresis,
Southern blotting, hybridizations, and autoradiography were conducted as
described elsewhere [26]. Probes were labeled with [32P]dCTP
by primer extension [27]. Following identification of
polymorphic RFLP markers (see Section 3 for rates of polymorphism), genomic DNAs of the 172 F2 individuals were digested with the 8 restriction enzymes and multiple sets of
Southern blots were prepared. Blots were hybridized with clones detecting
polymorphism and a total of 115 RFLP markers were scored in the F2 population.


## 2.3. EST Analysis

A set of unique ESTs was selected from the tomato gene index sources
maintained by The Institute for Genomic Research (TIGR;http://www.tigr.org/) (now at the Computational Biology and Functional
Genomics Laboratory at Harvard University; http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/est_report.pl). Each EST represents a valid (partial or complete)
copy of a transcribed functional allele. We selected 140 ESTs from a diverse
array of candidate genes and gene families, many of which are known or assumed
to play roles in disease-resistance or defense-response mechanisms. Among them
we included ESTs with homology to resistance (R) genes, signal transduction genes, transcriptional regulator factors,
and genes encoding pathogenesis-related proteins. We used this targeted
strategy to obtain a set of potentially functional markers for marker-assisted
selection in our tomato-breeding
program. The 140 EST clones, purchased from the
Clemson University Genomics Institute (http://www.genome.clemson.edu/), Clemson, SC, USA, were used as RFLP probes to identify
polymorphism between the two parents. Among them, 96 provided polymorphic
alleles (Table 1). The polymorphic ESTs were
used as RFLP probes to genotype the F2 individuals, examine their
segregation, and map onto the tomato chromosomes.Table 1
Listof ESTs mapped in theSolanum lycopersicum  ×  S. pimpinellifolium F2 population, their putative function,
chromosomal location, and copy
number.
EST cloneSaGN-TDPbutative functionChr.Ccopy no.cTOF3A14C146883Cytosolic Cu, Zn Superoxide dismutase,S. lycopersicum12cTOE7J7aC139397Endo-1,4-beta-glucanase,S. lycopersicum16cLED27E12C19568Cold
acclimation protein WCOR413-like protein form,O. sativa12cTOE6F10C139034Lipoxygenase,S. lycopersicum15cLEG9N2C45935Subunit A
of ferredoxin-thioredoxin reductase,S.
tuberosum11cLES9N20C79709ASC1 (Alternaria stem canker resistance protein),S. lycopersicum11cLEC6O2C11013Polyamine
oxidase,A. thaliana11cTOF20P4C142906Carotenoid
cleavage dioxygenase 1-2,S.
lycopersicum15cLEZ11K12C98684Snakin2
precursor,S. lycopersicum11cTOA9E13C117653Squalene synthase,C.
annuum15cTOA9C11C117644Similar to
WRKY transcription factor Nt-SubD48,N.
tabacum21cLET10E15C79822Acidic 26kDa endochitinase precursor,S. lycopersicum21cTOF19J9C142319Phosphoribosylanthranilate
isomerase,A. thaliana21cLEY1K9C97179Pathogen-inducible
alpha-dioxygenase,N. attenuata24cLEW11E20C89000Resistance complex protein I2C-3,S. lycopersicum27cTOF16A9C141311Calmodulin 3 protein,S. lycopersicum39cLER17H16C71298Elicitor-inducible cytochrome P450,N. tabacum31cTOF18P1C142154Serine palmitoyltransferase,S. tuberosum33cLEX12O16C92852Ethylene response factor 5,S.lycopersicum36cTOE2F15C137984Catalase isozyme 1,S. lycopersicum31cTOF29J22C1454124-coumarate-coA ligase 1,S. tuberosum32cLEX10F20C92172Ethylene response factor 1,S. lycopersicum34cTOF14B17C141010Anthocyanin 5-O-glucosyltransferase,S. sogarandinum41cLED15E5C16128Shikimate kinase chloroplast precursor,S. lycopersicum41cLEN13D5C66215Chorismate synthase 1 precursor,S. lycopersicum44cTOS21D12C163577Similar to
heat shock factor,N. tabacum43cTOF10N11C140057Myo-inositol-1-phosphate synthase,S. lycopersicum44/5cLEW24M21C90911TMV
disease resistance protein-like protein,Cicer
arietinum42cLEW22D11bC903524-coumarate:coenzyme A ligase,N. tabacum410cLER5E19C73560Phospholipase
PLDb1,S. lycopersicum51cTOC2J14aC127676Disease resistance gene homolog Mi-copy1,S.lycopersicum59cTOC2J14bC127676Disease resistance gene homolog Mi-copy1,S.lycopersicum59cTOF26E9C144413Prf,S.
pimpinellifolium52cTOE1K1C136851Spermidine synthase,S. lycopersicum54cTOE7J7bC139397Endo-1,4-beta-glucanase,S. lycopersicum56cTOF29B13C145236Metallothionein-like
protein type 2 a,S. lycopersicum52cTOF33C3C146601Serine/threonine
protein kinase Pto,S. lycopersicum510cTOF23J19C143585Heat shock
protein 90,S. lycopersicum54cLEG32E10C34795Lipoxygenase B,S.
lycopersicum66cTOF8F19C148467Ascorbate peroxidase,S. lycopersicum62cLEZ16H16C99197Contains
similarity to disease resistance response protein,Pisum sativum61cLED11A2C15134Mitogen-activated
protein (MAP) kinase 3,C. annuum62cLEW22D11aC903524-coumarate:coenzyme A ligase,N. Tabacum610cLEY21L21C97473Disease
resistance gene homolog Mi-copy1,S. lycopersicum66cLEW22N22C90504Ethylene-responsive
element binding factor 6-N. sylvestris63cTOF34C13C146804Peroxiredoxin
Q-like protein,A. thaliana71cLEN14F9C66474Sucrose-phosphate
synthase,S. lycopersicum71cTOF21F12C142982Dehydroquinate dehydratase/shikimate, NADP
oxidoreductase,S. lycopersicum79cLEN13G22C662461-aminocyclopropane-1-carboxylate oxidase,S. lycopersicum74cLEY22L20C97674Peroxidase precursor,S. lycopersicum73cTOE15M9C136013MYB-related
transcription factor VlMYBB1-1,Vitis
labrusca  ×  V. vinifera76cLEG34O20C35423UDP-glucose:salicylic acid glucosyltransferase,N. tabacum74cLEN14C8C66419PR-related protein, PR P23 (salt-induced protein),S. lycopersicum83cTOF9D16C148734Pathogenesis-related
protein 5-1,S. lycopersicum81cTOF28D12C144993Polyphenol oxidase E, chloroplast precursor,S.
lycopersicum87cLEN10H3C65539Heat shock
factor protein HSF8 (Heat shock transcription factor 8),S. lycopersicum82cLEI16E21C47449Cold-induced glucosyl transferase,S. lycopersicum83cTOF2N15C145786Osmotin-like protein OSML13 precursor (PA13),S. lycopersicum83cTOE23J12C137767Monodehydroascorbate reductase,S. lycopersicum83cLED27C20C19537DNADPH oxidase; gp91-phox homolog,S. lycopersicum81cLER14J12C70373WRKY
transcription factor IId-1 splice variant 2,S. lycopersicum81cTOF2L16C145747Phenylalanine ammonia-lyase (PAL),S. lycopersicum81cTOD3N7C132799Endo-1,4-beta-glucanase,S. lycopersicum82cLEX11E19C92435Putative NADH-ubiquinone oxireductase,A. thaliana91cTOE10J18C134749PR protein sth-2,S. Tuberosum93cLEC13E21C1592P14 (PR-Protein),S. lycopersicum93cLEC6M14C10964PR-protein sth-2,S. Tuberosum95cLER14J6C70387Hexose transporter,S.
lycopersicum91cTOF19O3C142383Hydroxyproline-rich
glycoprotein homolog,A. thaliana93cLEZ6E21bC100278Ubiquitin,S. lycopersicum104/5cLED18G6C17041Similar to
WRKY-like drought-induced protein,Retama
raetam106cTOD4I20C133021Tyrosine aminotransferase,A. thaliana102cLHT11J12C100975Diacylglycerol kinase,S. lycopersicum102cLER4F5C73337Ferredoxin-I chloroplast precursorS. lycopersicum104cTOF30K21C146034Chloroplast ferredoxin I,S. lycopersicum10>10cTOF22M16C143336NADH-ubiquinone oxidoreductase 23 kDa subunit,S. lycopersicum101cLEN14K6C66563Multiresistance protein homolog,A. thaliana103/2cLEX10N16C92314PR protein,S.
lycopersicum10>10cLEC18O1C3034Basic 30kDa endochitinase precursor,S. lycopersicum10>10cTOF31H10C146231Catechol O-methyltransferase,N. tabacum108cLEN9P2C69374Multiresistance
protein homolog,A. thaliana102cLED13I7C15652Resistance complex protein I2C-1,S. lycopersicum117cTOF28I23C145097Resistance
complex protein I2C-5,S.
pimpinellifolium11>10cLEZ6E21aC100278Ubiquitin,S. lycopersicum114/5cTOF29F6bC14533010-hydroxygeraniol oxidoreductase, -S. lycopersicum117cLEC14I18aC1998Resistance complex protein I2C-2,S. lycopersicum11>10cLEC14I18bC1998Resistance complex protein I2C-2,S. lycopersicum11>10cLEM22K17C627089-cis-epoxycarotenoid dioxygenase,S. lycopersicum117cLED23K21C18512Resistance complex protein I2C-5,S. lycopersicum11>10cLES18N16C76694Phosphatidylinositol 4-kinase,S. tuberosum112cTOS21D14C163579WRKY
transcription factor IId-2,S. lycopersicum121cLPT1G11C109877S-adenosyl-l-homocysteine
hydrolase,S. lycopersicum124cLEZ15E8C98979Extensin class I,S. Lycopersicum12>10cLEW25D9C90989Glutamine synthetase,S. lycopersicum123Saolanaceae Genome Network (SGN) can be accessed at http://www.sgn.cornell.edu/.Tbhe putative function of each EST has been derived
from Computational Biology and Functional Genomics Laboratory web site (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/est_report.pl), used to be maintained at The Institute for Genomic
Research (TIGR). (Computational Biology and
Functional Genomics Laboratory).Tche exact or approximate copy number of ESTs in tomato
genome was determined based on the number of hybridized bands on Southern blot
gels and may be varied in different labs. Where there is a “/” sign, the
figures in the left side denote the number of copies in S. lycopersicum parent and those in the right side denote the
number of copies in S. pimpinellifolium parent.


## 2.4. RGA Analysis

### 2.4.1. Selection of Primers

Ten pairs of
oligonucleotide primers, previously designed based on conserved LRR, NBS, and
PtoKin motifs of several resistance genes, were used (Table2; [28]). Some primers were chosen to be degenerate at the
redundant third position (3′ end) in the codons to cover a range of possible
sequences encoding the motifs, and thus to increase the efficiency of PCR
amplification [19, 29]. Only one pair of primers was
used for each PCR amplification.Table 2
Oligonucleotide primers
designed based on the conserved amino acid sequences within theLRR, NBS, and Pto protein domains encoded by
various R-genes.
GroupPrimersSequences (5′-3′)aDesign basisReferencesLRRCLRR forTTTTCGTGTTCAACGACGLRR domain of the tomatoCf-9 gene conferring resistance to Cladosporium
fulvum[30]CLRR revTAACGTCTATCGACTTCTNLRR forTAGGGCCTCTTGCATCGTLRR domain of the tobaccoN gene conferring resistance to TMVNLRR revTATAAAAAGTGCCGGACTRLRR forCGCAACCACTAGAGTAACLRR domain in theRPS2 gene conferring resistance to Pseudomonas syringae in ArabidopsisRLRR revACACTGGTCCATGAGGTTXLRR forCCGTTGGACAGGAAGGAGLRR domain of the riceXa21 gene conferring resistance to Xanthomonas campestris pv oryzaeXLRR revCCCATAGACCGGACTGTTANo. 2TATAGCGGCCGCIARIGCIARIGGIARNCCConserved P-loop and hydrophobic
NBS regions of theN and RPS2 genes from tobacco and Arabidopsis respectively[29]ANo. 3ATATGCGGCCGCGGIGGIGTIGGIAARACNACNBSS1GGTGGGGTTGGGAAGACAACGHydrophobic domain and P-loop of
conserved NBSs from theN and RPS2 genes from Arabidopsis and the L6 gene from flax conferring resistance to rust[18, 31]S2GGIGGIGTIGGIAAIACIACAS1CAACGCTAGTGGCAATCCAS3IAGIGCIAGIGGIAGICCPtokin1GCATTGGAACAAGGTGAASerine/threonine protein kinase
domain of thePto gene conferring
resistance to the bacterial pathogen Pseudomonas
syringae pv tomato in tomato[30]Ptokin2AGGGGGACCACCACGTAGPtokin3TAGTTCGGACGTTTACATPtoKinPtokin4AGTGTCTTGTAGGGTATCRLK forGAYGTNAARCCIGARAASerine/threonine kinase sequence subdomains of the wheatLr10 gene conferring resistance to Puccinia recondita[20]RLK revTCYGGYGCRATRTANCCNGGITGICCaCode for mixed
bases: D  =  A/G/T; I  =  Inosine;
N  =  A/G/C/T; R  =  A/G;  Y  =  C/T.


### 2.4.2. PCR Amplification

PCR conditions for amplification of RGAs were
described elsewhere [11]. Briefly, each amplification was performed in a 25-μL
volume consisting of 300 μM
each of dATP, dCTP, dGTP, and dTTP, 5 mM of MgCl2, 1 unit of Taq DNA polymerase, 2.5 μL of 10X buffer (PCR Core system I; Promega,
Madison, Wis, USA), 2 μL
of each primer, and 40 ng of genomic DNA that was used as template. For control
reactions, the template was substituted with sterile, nuclease-free ddH2O.
All PCR mixtures were overlaid with mineral oil and carried out in a Perkin
Elmer DNA Thermal Cycler 480 (Perkin Elmer, Foster City, Calif, USA), programmed
for 4 minutes at 94°C for an initial denaturation, and 36 cycles of
1 minute at 94°C (DNA denaturation), 1 minute at 50°C
(primer annealing), and 1.5 minutes at 72°C (primer extension),
followed by a final 7-minute extension at 72°C.


### 2.4.3. Gel Electrophoresis and Silver Staining

Denaturing polyacrylamide gel
electrophoresis (PAGE) was used to separate and detect individual RGA bands [30]. Briefly, a denaturing gel (7 M Urea—6% polyacrylamide) was prepared in a
sequencing gel apparatus (420  ×  330  ×  0.4 mm; Fisher
Biotech, Springfield, NJ, USA) using Bind- and Repel-Silane (Promega). After
polymerization, the gel was prerun in 1X Tris-borate-EDTA (TBE) buffer for 30
minutes at 40 W (~1400 V) to reach a gel temperature of 50°C.
Twelve μL
of loading buffer (10 M Urea—0.08% xylene cyanole) were added to each 25 μL
amplified DNA sample and the mixture was denatured at 95°C for 5
minutes and immediately put on ice. After cleaning the gel-loading surface, a
0.4 mm-thick shark comb (Fisher
Biotech, Springfield, NJ, USA) was inserted into the gel. Subsequently,
7 μL
of each PCR-amplified sample were loaded. Each gel accommodated 60 DNA samples
and three DNA size markers (1 Kb, 100 bp, 50 bp). The gel was run at 35 W (~1350 V) for 3.5–4 hours. After
electrophoresis, the gel, fixed to the Bind-Silane surface of one glass plate,
was silver stained following the manufacturer’s protocol (Promega). The gel was
air dried at room temperature overnight and stored in dark for future scoring
and scanning. All amplifications and gel electrophoresis procedures were
repeated at least once.


### 2.4.4. Identification of Informative RGA Markers

Following gel
electrophoresis and staining, polymorphic and monomorphic bands were observed.
Polymorphic bands were directly scored as dominant markers and used for genetic
mapping. To determine whether monomorphic bands could detect polymorphism if
used as RFLP probes, they were excised from the gel (as described
in [28, 32]), purified with the QIAgene quick
Gel Extraction Kit (QIAGEN, Valencia, Calif, USA), labeled with 32P-dCTP,
and used to hybridize the parental survey blots. Probes which detected
polymorphism between the two parents were then used to hybridize Southern blots
of the F2 population, and scored as either dominant or codominant
markers. Overall, a total of 43 RGA markers were successfully scored and mapped
onto the 12 tomato chromosomes.


### 2.4.5. Size Determination of RGA Fragments

PAGE polymorphic and monomorphic
fragments were excised from the dried polyacrylamide gel and reamplified, by
using a needle scratching and PCR reamplification method [32]. The reamplified products and DNA size markers (1 Kb, 100 bp, and 50 bp) were run on a 1.0% agarose gel, stained with ethidium
bromide, and photographed to determine the size.


## 2.4.1. Selection of Primers

Ten pairs of
oligonucleotide primers, previously designed based on conserved LRR, NBS, and
PtoKin motifs of several resistance genes, were used (Table2; [28]). Some primers were chosen to be degenerate at the
redundant third position (3′ end) in the codons to cover a range of possible
sequences encoding the motifs, and thus to increase the efficiency of PCR
amplification [19, 29]. Only one pair of primers was
used for each PCR amplification.Table 2
Oligonucleotide primers
designed based on the conserved amino acid sequences within theLRR, NBS, and Pto protein domains encoded by
various R-genes.
GroupPrimersSequences (5′-3′)aDesign basisReferencesLRRCLRR forTTTTCGTGTTCAACGACGLRR domain of the tomatoCf-9 gene conferring resistance to Cladosporium
fulvum[30]CLRR revTAACGTCTATCGACTTCTNLRR forTAGGGCCTCTTGCATCGTLRR domain of the tobaccoN gene conferring resistance to TMVNLRR revTATAAAAAGTGCCGGACTRLRR forCGCAACCACTAGAGTAACLRR domain in theRPS2 gene conferring resistance to Pseudomonas syringae in ArabidopsisRLRR revACACTGGTCCATGAGGTTXLRR forCCGTTGGACAGGAAGGAGLRR domain of the riceXa21 gene conferring resistance to Xanthomonas campestris pv oryzaeXLRR revCCCATAGACCGGACTGTTANo. 2TATAGCGGCCGCIARIGCIARIGGIARNCCConserved P-loop and hydrophobic
NBS regions of theN and RPS2 genes from tobacco and Arabidopsis respectively[29]ANo. 3ATATGCGGCCGCGGIGGIGTIGGIAARACNACNBSS1GGTGGGGTTGGGAAGACAACGHydrophobic domain and P-loop of
conserved NBSs from theN and RPS2 genes from Arabidopsis and the L6 gene from flax conferring resistance to rust[18, 31]S2GGIGGIGTIGGIAAIACIACAS1CAACGCTAGTGGCAATCCAS3IAGIGCIAGIGGIAGICCPtokin1GCATTGGAACAAGGTGAASerine/threonine protein kinase
domain of thePto gene conferring
resistance to the bacterial pathogen Pseudomonas
syringae pv tomato in tomato[30]Ptokin2AGGGGGACCACCACGTAGPtokin3TAGTTCGGACGTTTACATPtoKinPtokin4AGTGTCTTGTAGGGTATCRLK forGAYGTNAARCCIGARAASerine/threonine kinase sequence subdomains of the wheatLr10 gene conferring resistance to Puccinia recondita[20]RLK revTCYGGYGCRATRTANCCNGGITGICCaCode for mixed
bases: D  =  A/G/T; I  =  Inosine;
N  =  A/G/C/T; R  =  A/G;  Y  =  C/T.


## 2.4.2. PCR Amplification

PCR conditions for amplification of RGAs were
described elsewhere [11]. Briefly, each amplification was performed in a 25-μL
volume consisting of 300 μM
each of dATP, dCTP, dGTP, and dTTP, 5 mM of MgCl2, 1 unit of Taq DNA polymerase, 2.5 μL of 10X buffer (PCR Core system I; Promega,
Madison, Wis, USA), 2 μL
of each primer, and 40 ng of genomic DNA that was used as template. For control
reactions, the template was substituted with sterile, nuclease-free ddH2O.
All PCR mixtures were overlaid with mineral oil and carried out in a Perkin
Elmer DNA Thermal Cycler 480 (Perkin Elmer, Foster City, Calif, USA), programmed
for 4 minutes at 94°C for an initial denaturation, and 36 cycles of
1 minute at 94°C (DNA denaturation), 1 minute at 50°C
(primer annealing), and 1.5 minutes at 72°C (primer extension),
followed by a final 7-minute extension at 72°C.


## 2.4.3. Gel Electrophoresis and Silver Staining

Denaturing polyacrylamide gel
electrophoresis (PAGE) was used to separate and detect individual RGA bands [30]. Briefly, a denaturing gel (7 M Urea—6% polyacrylamide) was prepared in a
sequencing gel apparatus (420  ×  330  ×  0.4 mm; Fisher
Biotech, Springfield, NJ, USA) using Bind- and Repel-Silane (Promega). After
polymerization, the gel was prerun in 1X Tris-borate-EDTA (TBE) buffer for 30
minutes at 40 W (~1400 V) to reach a gel temperature of 50°C.
Twelve μL
of loading buffer (10 M Urea—0.08% xylene cyanole) were added to each 25 μL
amplified DNA sample and the mixture was denatured at 95°C for 5
minutes and immediately put on ice. After cleaning the gel-loading surface, a
0.4 mm-thick shark comb (Fisher
Biotech, Springfield, NJ, USA) was inserted into the gel. Subsequently,
7 μL
of each PCR-amplified sample were loaded. Each gel accommodated 60 DNA samples
and three DNA size markers (1 Kb, 100 bp, 50 bp). The gel was run at 35 W (~1350 V) for 3.5–4 hours. After
electrophoresis, the gel, fixed to the Bind-Silane surface of one glass plate,
was silver stained following the manufacturer’s protocol (Promega). The gel was
air dried at room temperature overnight and stored in dark for future scoring
and scanning. All amplifications and gel electrophoresis procedures were
repeated at least once.


## 2.4.4. Identification of Informative RGA Markers

Following gel
electrophoresis and staining, polymorphic and monomorphic bands were observed.
Polymorphic bands were directly scored as dominant markers and used for genetic
mapping. To determine whether monomorphic bands could detect polymorphism if
used as RFLP probes, they were excised from the gel (as described
in [28, 32]), purified with the QIAgene quick
Gel Extraction Kit (QIAGEN, Valencia, Calif, USA), labeled with 32P-dCTP,
and used to hybridize the parental survey blots. Probes which detected
polymorphism between the two parents were then used to hybridize Southern blots
of the F2 population, and scored as either dominant or codominant
markers. Overall, a total of 43 RGA markers were successfully scored and mapped
onto the 12 tomato chromosomes.


## 2.4.5. Size Determination of RGA Fragments

PAGE polymorphic and monomorphic
fragments were excised from the dried polyacrylamide gel and reamplified, by
using a needle scratching and PCR reamplification method [32]. The reamplified products and DNA size markers (1 Kb, 100 bp, and 50 bp) were run on a 1.0% agarose gel, stained with ethidium
bromide, and photographed to determine the size.


## 2.5. Statistical and Mapping Analyses

Segregation of the 250 DNA markers (115 RFLPs, 94 ESTs, and 41 RGAs) in the F2 population was tested for deviation from the expected Mendelian genotypic
ratios of 1:2:1 (for codominant) or 1:1 (for dominant markers) using chi-square
(χ2)
goodness-of-fit analysis. Multipoint linkage
analysis of the genetic markers in the F2 population was performed
using the MapMaker program v. 3.0 [33] and a genetic linkage map was
constructed. Briefly, the group command was used to assign markers into linkage
groups using a minimum LOD score of 3.0 and a maximum recombination fraction of
0.20. Three-point linkage analysis was performed to determine the maximum
likelihood recombination fraction and the associated LOD score for each
combination of loci. The “order” and “compare” commands
were used to find the best order of loci within each group, followed by using
the “ripple” command to verify the order. Markers were included
within the framework map only if the LOD value for the ripple was greater than
3.0. Once the linear order of markers along each chromosome was determined,
recombination frequencies between markers were estimated with multipoint
linkage analyses. The Kosambi mapping function [34] was used to convert
recombination frequencies to map distances in cM. The distribution of
percentage of the S. lycopersicum genome (L) in the F2 population was estimated using the computer program Qgene [35].


## 3. Results and Discussion

### 3.1. RFLP Polymorphism betweenS. Lycopersicum and S. Pimpinellifolium

RFLP clones
were chosen from two sources, a previously publishedS. lycopersicum (NC84173)  ×  S. pimpinellifolium (LA722) linkage map
(L  ×  PM2) [7] and the high-density S. lycopersicum (VF36 -Tm2a)  ×  S.
pennellii (LA716) linkage map of tomato (L  ×  P) [25]. Of the 152 RFLP clones chosen
from the L  ×  PM2 map, 82 (54%) were polymorphic between the
two parents (NCEBR-1 and LA2093) in the present study. Of the 120 clones that
were chosen based on the high-density L  ×  P map,
40 (30%) were polymorphic between NCEBR-1 and LA2093. The latter level of
polymorphism was similar to those previously reported by Grandillo and Tanksley [6] and Chen and Foolad [7] for different S. lycopersicum  ×  S. pimpinellifolium crosses. A lower
level of DNA polymorphism between S.
lycopersicum and S. pimpinellifolium compared
to that between S. lycopersicum and S. pennellii was expected as S. pimpinellifolium is phylogenetically
much closer to the cultivated tomato [1, 36, 37]. The high-density map of tomato
was constructed based on A S.
lycopersicum  ×  S.
pennellii cross mainly because of the presence of high level of marker
polymorphism between the two species. However, identification of polymorphic
markers and development of maps based S.
lycopersicum  ×  S. pimpinellifolium crosses are essential to facilitate marker-assisted exploitation of genetic
variation present in S. pimpinellifolium.
Such information may also be useful for exploitation of intraspecific variation
within S. lycopersicum. This is
because of frequent introgressions from S.
pimpinellifolium into the cultivated tomato, which have occurred both naturally and deliberately via plant
breeding [8]. In the present study, a total
of 117 polymorphic RFLP clones were used to construct the backbone linkage map.


### 3.2. EST Polymorphism betweenS. Lycopersicum and S. Pimpinellifolium

From
a total of 140 tomato ESTs examined, 91 (65%) were polymorphic between the two
parents. Five of 91 EST clones produced more than one polymorphic band, thus
resulting in the detection of a total of 96 polymorphic EST loci, including 91
codominant (~95%) and 5 dominant markers. Of the 96 EST markers, 94 were
successfully scored in the F2 population and mapped onto the 12
tomato chromosomes using the 115 RFLP anchor markers. The number of EST markers
per chromosome ranged from 4 (on chr. 12) to 12 (on chr. 10). Observation of a
high level of polymorphism in EST markers between S. lycopersicum and S. pimpinellifolium was unexpected, but
encouraging. This high level of polymorphism could be due to various reasons
including high copy number of EST bands (compared to the often single-copy RFLP
markers) and the nature of the genes or gene families from which ESTs were
selected. As indicated earlier, most ESTs were chosen based on their sequence
similarities with genes or proteins related to disease resistance. It is likely
that chromosomal regions containing resistance gene families accumulate a great
deal of variation during their evolution, thus increasing the frequency of
restriction sites, which are a basis for polymorphism. Because modern breeding
lines have received frequent introgressions from different tomato wild species,
in particular for disease resistance, presence of such introgressions in
NCEBR-1 could have contributed to the high level of observed polymorphism.
Further inspections of the chromosomal locations of ESTs support this
submission, as discussed below. However, the observation of high level of EST
polymorphism is promising as larger number of ESTs are becoming available.


### 3.3. Marker Segregation

Of
the 250 marker loci scored in the L  ×  PM3 F2 population, 41 (16.4%) exhibited significant deviation from the expected 1:2:1
(codominant) or 1:1 (dominant) segregation ratios at P≤.01.
Markers with skewed segregation were located on chromosomes 1, 3, 4, 5, and 6,
with those on chromosome 6 exhibiting the highest level of skewness (Table 3). Markers on chromosomes 1, 3, and 4
exhibited distortion in favor of S.
pimpinellifolium alleles whereas those on chromosomes 5 and 6 were in favor
of S. lycopersicum. Observation of
extensive segregation distortion for markers on chromosome 6 was not unexpected
and could be attributed to the selection of determinate F2 plants
(as described in Section 2) and the presence of self-pruning (sp) locus on this chromosome (~3 cM from RFLP marker TG279) [6]. Skewed segregation for markers
on this chromosome was previously reported in other interspecific crosses of
tomato, where phenotypic selection (PS) or MAS was employed to remove
indeterminate plants from mapping populations [28, 38, 39]. However, in the present study,
despite skewed segregation for markers on chromosome 6, no major differences in
genetic map distances were observed when they were compared with the
high-density map of tomato [39] or the previous S. lycopersicum  ×  S. pimpinellifolium maps [6, 7], where no such selections were
practiced.Table 3
Significant deviations from the expected 3:1 and 1:1 ratios
in theSolanum
lycopersicum  ×  S. pimpinellifolium F2 population (L: lycopersicum allele, PM: pimpinellifolium allele).
LocusChromosomeGenotypeL/LL/PMPM/PMPM/−L/−χ2*AN23_24011600141018.36S13_31011600139017.81S11_18011600140018.09S11_15011700139016.55S11_20011700139016.55NBS4_30011878400010.06TG12511884430012.27cTOF3A1412091430011.96TG1323288557009.89TG663229044009.90CT225B31591340017.76cLEX10F2032497360010.55CT8232498380010.55cLER17H163566932009.64cLEW24M214366355009.78CT17843574600010.01C254356756009.23CT734426758009.59CT9355198170019.35cLER5E1950016013817.53TG50355087170016.74cTOF33C354586140018.28cTOF26E9551100160021.19TG9655079140019.70cTOF23J1953582130016.34XLRR38050014014321.66TG35154487180013.27cTOC2J14a53499120026.05cTOC2J14b55979130028.35cTOF29B1355978140026.99TG18554675120019.56CT28566172270016.05TG35668253160071.11cLEW22D11a692441300108.74cLEW22N22610339600160.26TG365611841700190.95TG253613230400265.08C54615411200402.59TG27961564100443.84cLEZ16H16614222100329.72TG477613524100302.85*All χ2 values  significant at P<.01.Skewed segregation has been reported in many interspecific
crosses of tomato, with the extent of skewness being greater in wider crosses
compared to crosses between closely related species, and also generally greater
in F2 than in backcross populations [6, 40–45]. A survey of recently published
results of interspecific crosses of tomato indicated that skewed segregation
was 8.3% in the L  ×  PM1 BC1 population [6], 9.9% in the L  ×  PM2 BC1 population [7], 51% in a S. lycopersicum  ×  S.
cheesmaniae (L  ×  CH) F2 population [42], 69% in a S. lycopersicum  ×  S.
chmielewskii (L  ×  CL) BC1 population [46], 15% in a S. lycopersicum  ×  S.
habrochaites (L  ×  H1) BC1 population [38], 62% in the L  ×  H2 BC1 population [28], and
80% in a S. lycopersicum  ×  S.
pennellii (L  ×  P) F2 population [47]. The L  ×  PM populations
exhibited less overall skewed segregation than the other interspecific crosses,
consistent with the close phylogenetic relationship between S. lycopersicum and S. pimpinellifolium. However, the relatively high level of skewed
segregation in the L  ×  CH F2 population [42] and the low level of skewed
segregation in the L  ×  H1 BC1 populations [38] were unordinary because S. cheesmaniae is a closely related and S. habrochaites is a distantly related
wild species of tomato [1, 9, 10, 48, 49]. Skewed
segregation in interspecific crosses of tomato has been attributed to various
causes, including self-incompatibility (SI), unilateral incongruity, and
gametophytic, zygotic, and viability selection in segregating populations, as
discussed elsewhere [44, 50–52].


### 3.4. Genome Composition of theF2 Population

The
genomic compositions of the 172 F2 individuals were determined based
on the 220 codominant markers using qgene
program. On average, the F2 population was inferred to
contain 51.5% of its genome from the S.
lycopersicum parent (L alleles), which is very close to the expected 50%.
The percent L genome of individual F2 plants ranged from 41.4% to
97.8% (Figure 1), indicating the high level of variation in the F2 population. This analysis clearly demonstrates the power of marker genotyping
for precise determination of the genomic composition of individual plants in
breeding populations. Such information can facilitate the selection of suitable
plants and introgression of desirable and elimination of undesirable
chromosomal segments in genetic populations derived via backcross breeding. For
example, in the present population, individuals with ≥65% L genome (Figure 1) could be returned to nearly 100% L
genome within 2–4 backcrosses,
far more rapid than the 4–6 backcrosses
routinely needed to eliminate donor genome without MAS. Alternatively, in a
pedigree-type breeding program, marker analysis (if economically feasible) can
facilitate inbreeding to homozygosity by selecting progeny at each generation
which are homozygous
over a maximal proportion of the genome.Figure 1
Distribution of percentSolanum lycopersicum genome in the F2  population, estimated based on 220 codominant markers.


### 3.5. Construction of the Linkage Map

A genetic
linkage map was constructed based on 115 RFLP, 94 EST, and 41 RGA loci using the F2 population of 172 individuals. The present map (L  ×  PM3) spanned 1002.4 cM of
tomato genome with an average marker interval length of 4.0 cM (Figure 2). The number of markers per chromosome
ranged from 16 (chrs. 3 and 7) to 28 (chr. 1). Chromosome 1 had the largest
linkage group (102.9 cM) followed by chromosomes 9 and 2 (96.1 and 92.6 cM, resp.),
whereas chromosome 7 had the smallest one (69.8 cM), preceded by chromosomes 4
and 5 (72.2 and 70.6 cM, resp.). Only two regions, on chromosomes 3 and 12,
contained marker intervals larger than 20 cM (Figure 2), and this was mainly
because of the low level of polymorphism between the two parents of this
mapping population for markers on these chromosomes. This map was compared with
several other molecular linkage maps of tomato for marker order, recombination
frequencies, and total map length, as described below.Figure 2
A genetic
linkage map of tomato constructed based on an F2 population of a cross between a tomato (S. lycopersicum)
breeding line (NCEBR-1) and an accession (LA2093) of tomato wild species S. pimpinellifolium and 250 RFLP, EST,
and RGA markers. RFLP markers are shown in blue, ESTs in green, and RGAs in red
fonts. The names of the markers are shown at the right and the map distances
between them (in cM, using Kosambi function) are shown at the left of the
chromosomes. The approximate chromosomal locations of disease-resistance genes
(R-genes) and quantitative resistance loci (QRL), as inferred from other
published researches, are shown in parentheses to the right of chromosomes. The
descriptions of the R-genes and QRL are as follows: Asc: resistance to Alternaria stem canker (Alternaria alternata f. sp. lycopersici)
[54, 55]; Bw (1–5) or Rrs (3–12): QLRs for
resistance to bacterial wilt (Ralstonia
solanacearum) [56–59]; Cf (1–9, ECP2):
resistance to leaf mould (Cladosporium
fulvum) [60–65]; Cmr: cucumber mosaic virus [66]; Fen: sensitivity to herbicide fenthion [67]; Frl: resistance to Fusarium crown and root rot (Fusarium oxysporum f. sp.
radicis-lycopersici) [68]; Hero: resistance to potato cyst namatode (Globodera rostochiensis) [69]; I (I,1, 2, 2C, 3): resistance to different
races of Fusarium wild (Fusarium
oxysporum f. sp. lycopersici) [70–78]; Lv: resistance to powdery mildew (Leveuillula taurica) [79]; Meu-1: resistance to potato aphid [80–82]; Mi (Mi, 1, 2, 3, 9):
resistance to root knot nematodes (Meloidogyne spp.) [81, 83–89]; Ol (1, 2, 3): resistance
to powdery mildew (Oidium lycopersicum)
[90, 91]; Ph (1, 2, 3): resistance
to late blight (Phytophthora infestans)
in tomato [92–94]; Pot-1: resistance to potyvirus [95]; Pto and Prf: resistance
to bacterial speck (Pseudomonase syringae pv tomato) [96, 97]; Py-1: resistance to corky root rot (Pyrenochaeta lycopersici) [98]; Rcm (1–10): QRL for
resistance to bacterial canker (Clavibacter
michiganensis) [99, 100]; Rrs (3–12) or Bw (1–5):
QLRs for resistance to bacterial wilt (Ralstonia
solanacearum) [56–59]; Rx (1, 2, 3, 4):
resistance to bacterial spot (Xanthomonas
campestris) [101–103]; Sm: resistance to Stemphilium [104]; Sw-5: resistance to tomato-spotted wilt virus [105, 106]; Tm-1 and Tm-2a:
resistance to tobacco mosaic virus [68, 107–110]; Ty (1, 2, 3): resistance
to tomato yellow leaf curl virus [111–113]; Ve: resistance to Verticillium dahliae [114, 115].


### 3.6. Mapping of ESTs

The use of the 115 RFLP anchor markers
facilitated mapping of the 94 EST loci onto the 12 tomato chromosomes. The
number of ESTs per chromosome ranged from 4 (chr. 12) to 12 (chr. 10) (Figure2). The use of ESTs as genetic markers has several advantages. First, they can
be used as codominant markers for genetic mapping and QTL identification [53]. Although ESTs were used as RFLP
markers, that is, through Southern hybridization, technically they can be
converted to PCR-based markers adapted to high-throughput analysis. Such
conversion may reduce polymorphism level, in particular between closely related
individuals, though it is expected to enhance their utility as genetic markers.
Second, mapping of ESTs can facilitate association of functionality with
phenotype. EST markers are derived from partial or complete sequences of cDNA
clones, which may provide information on gene function. Third, coding
sequences, especially those of house-keeping genes, are rather conserved across
species. Mapping of ESTs and comparative genomics may lead to the detection of
new genes in different species.Inspections of the distribution of ESTs on different
chromosomes indicated that in some cases they were clustered, for example, ESTs
on chromosomes 4, 8, 10, and 11. Further inspections indicated that chromosomal
locations of some clustered or individual ESTs were colocalized with
approximate locations of some major disease-resistance genes (R-genes) or
quantitative resistance loci (QRLs), as inferred from other published researche (see Figure 2). While such colocalization suggests that these ESTs may be genetically
related to resistance genes or QRL, their actual functionality relationships
can only be determined by further analyses such as isolation and sequencing of
full EST sequences and functional genomic studies.Currently, there are more than 214 000 ESTs identified in
tomato (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=tomato), of which
only a small percentage has been mapped onto the tomato chromosomes (http://www.sgn.cornell.edu/cgi-bin/search/direct_search.pl?search=EST). The ESTs
were derived from more than 23 cDNA libraries [116, 117] and their sequences are
available on Solanaceae Genome Network (SGN; http://www.sgn.cornell.edu/). All but
four (cLET10E15, cLER4F5, cLEC6O2, and cLEG9N2) of the ESTs mapped in the
present study were not previously mapped onto tomato chromosomes. Moreover, of
the four that were previously mapped, different members of the corresponding
contigs were mapped onto the same or different tomato chromosomes as in the
present study. For example, cLET10E15 and cLER4F5 have overlap sequences with
cLET1A5 and cLET3F16, respectively, and were mapped on the same chromosomes (http://www.sgn.cornell.edu/) as in the
present study. cLEC6O2, which was mapped to chromosome 1 in the present study,
was mapped to chromosome 8 and named cLPT1J10 (SGN: F2 population ofa cross S. lycopersicum LA925  ×  S. pennellii LA716). EST
clone cLEG9N2, which was mapped to chromosome 1 in this study, was previously
mapped under cLET20B4 but with no known chromosomal position (http://www.sgn.cornell.edu/). Also, as
indicated earlier, five of the EST clones resulted in two pairs of polymorphic
bands each. For two of these clones, the two polymorphic bands were mapped onto
two linked loci, that is, cTOC2J14a and cTOC2J14b on chromosome 5 and
cLEC14I18a and cLEC14I18b on chromosome 11. Others
were mapped onto different chromosomes; for example, cLEW22D11a was mapped to
chromosome 6 whereas cLEW22D11b to chromosome 4, and cTOE7J7a was mapped to
chromosome 1 whereas cTOE7J7b to chromosome 5.


### 3.7. Mapping of RGAs

PCR
amplification using the 10 pairs of RGA primers (Table2) followed by
denaturing PAGE resulted in the detection of a few hundred polymorphic and
monomorphic bands. As described in Section 2, of the detected bands, 41 were
strong and verifiable and thus were scored in the F2 population. The
amplified fragment size of these RGA bands ranged from 150 to 760 bp. Linkage
analysis indicated that the 41 RGA markers were located on the 12 tomato
chromosomes, ranging in number from 1 (on chrs. 3, 5, and 7) to 9 (on chr. 1)
(Figure 2). The results indicated that RGA loci could be used as genetic
markers for genome mapping, consistent with previous suggestions [28, 30]. In several cases, RGA loci were
clustered, similar to that observed for R-genes
in various plant species [17, 19, 29, 60, 118–120]. For example, on each of
chromosomes 1, 2, 9, 10, 11, and 12, three or more RGA loci that were amplified
from the same or different primer pairs mapped to the same or nearby positions
(Figure 2). This observation indicated that different primers might initiate
amplification of closely linked RGA loci that might be members of the same or
different gene families.Map positions of RGA loci were compared with chromosomal
positions of known tomatoR-genes and major QRL, whose positions were inferred from the previously published maps, as displayed and
described in Figure 2. Most positions were inferred based on linkage to
reference markers and thus should be considered best approximations. Colocalization of RGA loci with R-genes and QRLs were observed on a few
chromosomes, including regions on chromosomes 1, 2, 7, 8, 9, 10, 11, and 12
(Figure 2). These observations suggest the possibility of the presence of R or
DR genes at the locations of RGAs, though this hypothesis could be confirmed
only by extensive mapping and functional analysis of RGAs. Specifically,
mapping of the associated RGAs in populations segregating for the colocalized R-genes
and cloning and molecular characterization of RGAs are necessary before any
functional relationship could be established.The map positions of the RGA loci in the present map (L  ×  PM3) were compared with those reported in aS.
lycopersicum  ×  S.
habrochaites (L  ×  H2) map [28]. There were 19 common RGA loci
between the two populations and 13 (68%) of which mapped to the same locations
in the two maps, suggesting consistent and reproducible positions of RGAs
across populations.


### 3.8. Comparison of the Map with Other Molecular Linkage Maps of Tomato

The
present map (L  ×  PM3) was compared with two previously developedS. lycopersicum  ×  S. pimpinellifolium maps, including L  ×  PM1 [6] and L  ×  PM2 [7] as well as the high-density S. lycopersicum  ×  S. pennellii (L  ×  P) map of tomato [25]. The present map is different
but complementary to L  ×  PM1 and L  ×  PM2 maps in several ways. First, different S.
lycopersicum and S. pimpinellifolium parents
and pretty much different molecular markers were used in the construction of
the three maps. The L  ×  PM1 was constructed based on a cross between a processing
tomato cultivar (M82-1-7) and S.
pimpinellifolium accession LA1589 using ~120 RFLP and RAPD markers. The L  ×  PM2 was constructed based on a cross between a fresh market tomato breeding
line (NC84173) and S. pimpinellifolium accession
LA722 using 151 RFLP markers. The current map (L  ×  PM3) was constructed based
on 250 RFLP, EST, and RGA markers using superior parental lines, as described
earlier. It is expected that this map will have great utilities, including
exploitation of the genetic potential of LA2093 and other S. pimpinellifolium accessions.The second point of
difference is that relatively a small percentage of the markers used in the
present study were used in the previous twoS.
lycopersicum  ×  S.
pimpinellifolium linkage maps. Specifically, a new set of RFLP clones that
detect polymorphism between S.
lycopersicum and S. pimpinellifolium has
been identified in the present study, beyond those that were identified in the
construction of the previous two maps. However, an important observation is
that markers that are polymorphic in one L  ×  PM cross usually have a greater
chance of being polymorphic in other L  ×  PM crosses, compared to markers
directly chosen from the high-density L  ×  P map. Nonetheless, the observation
that only 54% of the mapped RFLP clones in the L  ×  PM2 population were
polymorphic in the L  ×  PM3 population indicates the presence of considerable
DNA sequence variation among S.
pimpinellifolium accessions. The overall results suggest that while for
each S. pimpinellifolium accession
new polymorphic markers need to be identified, the most useful sources would be
those markers that have already been mapped in other S. lycopersicum  ×  S.
pimpinellifolium crosses. Third, unlike in
the previous two L  ×  PM maps, in the present map, “functional” markers such as
ESTs and RGAs were used. Such markers may be more useful than random genetic
markers for identification of candidate genes. The use of a large number of
markers and the incorporation of functional markers in the present map extends
its practical value in various genetics and breeding studies. However, the
availability of three L  ×  PM maps with rather different molecular markers should
facilitate marker-assisted exploitation of these and other S. pimpinellifolium accessions.When the current map was compared with L  ×  PM1 [6], L  ×  PM2 [7], and the high-density L  ×  P map [25], it was determined that the
linear order of
the common markers were generally the same. However, there were differences in
interval lengths for several adjacent markers. For example, of 13 common marker
intervals between L  ×  PM3 and L  ×  PM2 maps, 6 intervals on chromosomes 2, 3, 5,
6, and 12 differed in length by 2-3 fold, of which 1
interval was expanded in L  ×  PM3 map. The difference between the two maps in
marker interval lengths was not unexpected given the use of different type populations (F2 versus BC1), rather small
size populations (172 and 119) and different number of markers (250 versus
151), all of which could have affected the occurrence and detection of
recombination in different intervals. When the L  ×  PM3 was compared with the
high-density L  ×  P map, which was constructed based on >1 000 genetic
markers and 67 F2 plants, genetic distances differed markedly for a
large number of marker intervals. For example, for 36 common marker intervals,
genetic distances differed between the two maps by at least twofold; of these,
7 intervals (23%) showed decreased and 13 (36%) showed increased recombination
in the L  ×  PM3 map. Greater differences in marker interval lengths between L  ×  PM3 and L  ×  P maps compared to that between L  ×  PM3 and L  ×  PM2 maps was not unusual
considering the relatively close phylogenetic relationships between the L  ×  PM3
and L  ×  PM2 mapping populations.When comparing the L  ×  PM3 map with the high-density L  ×  P
map, the most striking differences in genetic distances were observed in
centromeric regions of chromosomes 3, 4, and 9, where substantial expansions in
map distances were observed in the L  ×  PM3 map, and in two locations on
chromosome 12, where substantial contractions were observed in the L  ×  PM3 map
(Table4 and Figure 1). The
decrease in recombination frequencies in the centromeric regions of tomato
chromosomes was previously attributed to the centromeric suppression of
recombination [5, 121, 122]. Such suppression was suggested
to be more frequent in wider crosses than in intraspecific crosses and crosses
between closely related species. Further inspections indicated that the
differences in genetic distances between the two maps across the rest of the
genome were generally interval specific and not a characteristic of individual
chromosomes. For example, for chromosomes 2, 3, 6, 10, 11, and 12, the L  ×  PM3
map exhibited expansion in some intervals and contraction in others (Table 4).
As indicated earlier, such differences were due in part to the detection of
chance recombination given the limited population sizes used in these studies.Table 4
Comparison of map distances based on common marker
intervals between three molecular linkage maps of tomatoa.
IntervalChr.Marker interval map distance (cM)(L  ×  PM3)b(L  ×  PM2)c(L  ×  PM3)/(L  ×  PM2)(L  ×  P)d(L  ×  PM3)/(L  ×  P)TG70-TG273124.323.81.08.92.7*TG554-TG45326.6——0.0NA*TG453-TG14520.4——6.80.1*TG145-CT103210.3——10.11.0CT176-TG58225.618.90.3*15.50.4*CT59-TG62022.67.00.4*0.0NA*TG114-TG13230.08.9N/A*15.4NA*TG66-CT225B36.6——1.83.7*CT225B-CT8239.8——6.31.6CT82-TG515312.714.80.93.93.3*TG123-TG182412.5——14.20.9TG182-TG60946.7——5.51.2TG609-CT178411.8——11.11.1CT167-CT93519.38.92.2*12.91.5TG503-TG9652.5——3.20.8TG274-TG59064.6——10.40.4*TG356-TG365613.011.91.14.13.2*C54-TG27964.010.20.4*—NATG183-TG128715.7——2.36.8*TG128-CT22673.13.50.91.61.9TG128-TG174719.313.61.410.71.8TG176-CD4088.7——0.0NA*CT265-TG294812.89.61.313.11.0TG486-CD391.7——1.31.3CD3-CT279911.3——5.62.0*CT279-TG3591.9——0.0NATG408-CD34104.9——22.90.2*CD34-TG4031030.137.50.87.44.1*TG629-TG497110.0——0.0NATG30-CT651116.7——3.94.3*TG68-CT79123.36.70.5*14.40.2*CT99-TG618125.4——0.86.8*TG618-TG1111212.5——6.12.0*TG111-TG565123.0——0.0NA*CT156-TG473126.1——19.10.3*TG473-CD2120.0——1.80.0aOnly common
marker intervals that were different in length by at least twofold between L  ×  PM1 and either
L  ×  PM2 or L  ×  P linkage maps are shown.bL  ×  PM3: Solanum lycopersicum (NCEBR-1)  ×  S. pimpinellifolium (LA2093) map
(present map).cL  ×  PM2: S. lycopersicum (NC84173)  ×  S. pimpinellifolium (LA722) map
[7]
.dL  ×  P: S.lycopersicum (VF36-Tm2)  ×  S. pennellii (LA716) map
[25]
.*Difference
in interval length by at least twofold. Dashes (—) indicate
no common interval for comparison. NA indicates a number divided by 0.0, 0.0
over a number, or no comparison was made.Comparisons were also made across the four maps (L  ×  PM3, L  ×  PM2, L  ×  PM1, and L  ×  P) in terms of individual chromosome and total map
lengths. The total length of the current map (1002 cM) was comparable with that
of the L  ×  P (1277 cM), L  ×  PM1 (1275 cM), and the L  ×  PM2 (1186 cM) maps.
Furthermore, across the maps the length of each chromosome in the current map  was comparable to the corresponding chromosome in the other
maps (Table5).Table 5
Pairwise comparison of the present map (L × PM3) with other maps of tomato for individual chromosome lengths based on orthologous markers.
Chromosome length (cM)Linkage mapa123456789101112AverageTotalL × PM3102.992.685.372.270.674.669.886.696.180.688.383.483.61003.0L × PM2129.7121.9133.810894.182.891.364.4104.884.978.292.698.91186.5L × PM1149.698.2116.697.2108.285.2116.486.1104.2101.5107105.2106.31275.4L × P133.5124.2126.1124.897.4101.991.694.911190.18893.1106.41276.6L × PM3/L × PM20.80.80.60.70.80.90.81.30.90.91.10.90.8L × PM3/L × PM10.70.90.70.70.70.90.61.00.90.80.80.80.8L × PM3/L × P0.80.70.70.60.70.70.80.90.90.91.00.90.8*L × PM3, S. lycopersicum (NCEBR-1) × S. pimpinellifolium (PSLP125) map (the present map); L × PM2, S. lycopersicum (NC84173) × S. pimpinellifolium (LA722) map [7]; L × PM1, S. lycopersicum (M82-1-7) × S. pimpinellifolium (LA1589) map [6]; E × P, S. lycopersicum (VF36-Tm2) × S. pennellii (LA716) map [25].


## 3.1. RFLP Polymorphism betweenS. Lycopersicum and S. Pimpinellifolium

RFLP clones
were chosen from two sources, a previously publishedS. lycopersicum (NC84173)  ×  S. pimpinellifolium (LA722) linkage map
(L  ×  PM2) [7] and the high-density S. lycopersicum (VF36 -Tm2a)  ×  S.
pennellii (LA716) linkage map of tomato (L  ×  P) [25]. Of the 152 RFLP clones chosen
from the L  ×  PM2 map, 82 (54%) were polymorphic between the
two parents (NCEBR-1 and LA2093) in the present study. Of the 120 clones that
were chosen based on the high-density L  ×  P map,
40 (30%) were polymorphic between NCEBR-1 and LA2093. The latter level of
polymorphism was similar to those previously reported by Grandillo and Tanksley [6] and Chen and Foolad [7] for different S. lycopersicum  ×  S. pimpinellifolium crosses. A lower
level of DNA polymorphism between S.
lycopersicum and S. pimpinellifolium compared
to that between S. lycopersicum and S. pennellii was expected as S. pimpinellifolium is phylogenetically
much closer to the cultivated tomato [1, 36, 37]. The high-density map of tomato
was constructed based on A S.
lycopersicum  ×  S.
pennellii cross mainly because of the presence of high level of marker
polymorphism between the two species. However, identification of polymorphic
markers and development of maps based S.
lycopersicum  ×  S. pimpinellifolium crosses are essential to facilitate marker-assisted exploitation of genetic
variation present in S. pimpinellifolium.
Such information may also be useful for exploitation of intraspecific variation
within S. lycopersicum. This is
because of frequent introgressions from S.
pimpinellifolium into the cultivated tomato, which have occurred both naturally and deliberately via plant
breeding [8]. In the present study, a total
of 117 polymorphic RFLP clones were used to construct the backbone linkage map.


## 3.2. EST Polymorphism betweenS. Lycopersicum and S. Pimpinellifolium

From
a total of 140 tomato ESTs examined, 91 (65%) were polymorphic between the two
parents. Five of 91 EST clones produced more than one polymorphic band, thus
resulting in the detection of a total of 96 polymorphic EST loci, including 91
codominant (~95%) and 5 dominant markers. Of the 96 EST markers, 94 were
successfully scored in the F2 population and mapped onto the 12
tomato chromosomes using the 115 RFLP anchor markers. The number of EST markers
per chromosome ranged from 4 (on chr. 12) to 12 (on chr. 10). Observation of a
high level of polymorphism in EST markers between S. lycopersicum and S. pimpinellifolium was unexpected, but
encouraging. This high level of polymorphism could be due to various reasons
including high copy number of EST bands (compared to the often single-copy RFLP
markers) and the nature of the genes or gene families from which ESTs were
selected. As indicated earlier, most ESTs were chosen based on their sequence
similarities with genes or proteins related to disease resistance. It is likely
that chromosomal regions containing resistance gene families accumulate a great
deal of variation during their evolution, thus increasing the frequency of
restriction sites, which are a basis for polymorphism. Because modern breeding
lines have received frequent introgressions from different tomato wild species,
in particular for disease resistance, presence of such introgressions in
NCEBR-1 could have contributed to the high level of observed polymorphism.
Further inspections of the chromosomal locations of ESTs support this
submission, as discussed below. However, the observation of high level of EST
polymorphism is promising as larger number of ESTs are becoming available.


## 3.3. Marker Segregation

Of
the 250 marker loci scored in the L  ×  PM3 F2 population, 41 (16.4%) exhibited significant deviation from the expected 1:2:1
(codominant) or 1:1 (dominant) segregation ratios at P≤.01.
Markers with skewed segregation were located on chromosomes 1, 3, 4, 5, and 6,
with those on chromosome 6 exhibiting the highest level of skewness (Table 3). Markers on chromosomes 1, 3, and 4
exhibited distortion in favor of S.
pimpinellifolium alleles whereas those on chromosomes 5 and 6 were in favor
of S. lycopersicum. Observation of
extensive segregation distortion for markers on chromosome 6 was not unexpected
and could be attributed to the selection of determinate F2 plants
(as described in Section 2) and the presence of self-pruning (sp) locus on this chromosome (~3 cM from RFLP marker TG279) [6]. Skewed segregation for markers
on this chromosome was previously reported in other interspecific crosses of
tomato, where phenotypic selection (PS) or MAS was employed to remove
indeterminate plants from mapping populations [28, 38, 39]. However, in the present study,
despite skewed segregation for markers on chromosome 6, no major differences in
genetic map distances were observed when they were compared with the
high-density map of tomato [39] or the previous S. lycopersicum  ×  S. pimpinellifolium maps [6, 7], where no such selections were
practiced.Table 3
Significant deviations from the expected 3:1 and 1:1 ratios
in theSolanum
lycopersicum  ×  S. pimpinellifolium F2 population (L: lycopersicum allele, PM: pimpinellifolium allele).
LocusChromosomeGenotypeL/LL/PMPM/PMPM/−L/−χ2*AN23_24011600141018.36S13_31011600139017.81S11_18011600140018.09S11_15011700139016.55S11_20011700139016.55NBS4_30011878400010.06TG12511884430012.27cTOF3A1412091430011.96TG1323288557009.89TG663229044009.90CT225B31591340017.76cLEX10F2032497360010.55CT8232498380010.55cLER17H163566932009.64cLEW24M214366355009.78CT17843574600010.01C254356756009.23CT734426758009.59CT9355198170019.35cLER5E1950016013817.53TG50355087170016.74cTOF33C354586140018.28cTOF26E9551100160021.19TG9655079140019.70cTOF23J1953582130016.34XLRR38050014014321.66TG35154487180013.27cTOC2J14a53499120026.05cTOC2J14b55979130028.35cTOF29B1355978140026.99TG18554675120019.56CT28566172270016.05TG35668253160071.11cLEW22D11a692441300108.74cLEW22N22610339600160.26TG365611841700190.95TG253613230400265.08C54615411200402.59TG27961564100443.84cLEZ16H16614222100329.72TG477613524100302.85*All χ2 values  significant at P<.01.Skewed segregation has been reported in many interspecific
crosses of tomato, with the extent of skewness being greater in wider crosses
compared to crosses between closely related species, and also generally greater
in F2 than in backcross populations [6, 40–45]. A survey of recently published
results of interspecific crosses of tomato indicated that skewed segregation
was 8.3% in the L  ×  PM1 BC1 population [6], 9.9% in the L  ×  PM2 BC1 population [7], 51% in a S. lycopersicum  ×  S.
cheesmaniae (L  ×  CH) F2 population [42], 69% in a S. lycopersicum  ×  S.
chmielewskii (L  ×  CL) BC1 population [46], 15% in a S. lycopersicum  ×  S.
habrochaites (L  ×  H1) BC1 population [38], 62% in the L  ×  H2 BC1 population [28], and
80% in a S. lycopersicum  ×  S.
pennellii (L  ×  P) F2 population [47]. The L  ×  PM populations
exhibited less overall skewed segregation than the other interspecific crosses,
consistent with the close phylogenetic relationship between S. lycopersicum and S. pimpinellifolium. However, the relatively high level of skewed
segregation in the L  ×  CH F2 population [42] and the low level of skewed
segregation in the L  ×  H1 BC1 populations [38] were unordinary because S. cheesmaniae is a closely related and S. habrochaites is a distantly related
wild species of tomato [1, 9, 10, 48, 49]. Skewed
segregation in interspecific crosses of tomato has been attributed to various
causes, including self-incompatibility (SI), unilateral incongruity, and
gametophytic, zygotic, and viability selection in segregating populations, as
discussed elsewhere [44, 50–52].


## 3.4. Genome Composition of theF2 Population

The
genomic compositions of the 172 F2 individuals were determined based
on the 220 codominant markers using qgene
program. On average, the F2 population was inferred to
contain 51.5% of its genome from the S.
lycopersicum parent (L alleles), which is very close to the expected 50%.
The percent L genome of individual F2 plants ranged from 41.4% to
97.8% (Figure 1), indicating the high level of variation in the F2 population. This analysis clearly demonstrates the power of marker genotyping
for precise determination of the genomic composition of individual plants in
breeding populations. Such information can facilitate the selection of suitable
plants and introgression of desirable and elimination of undesirable
chromosomal segments in genetic populations derived via backcross breeding. For
example, in the present population, individuals with ≥65% L genome (Figure 1) could be returned to nearly 100% L
genome within 2–4 backcrosses,
far more rapid than the 4–6 backcrosses
routinely needed to eliminate donor genome without MAS. Alternatively, in a
pedigree-type breeding program, marker analysis (if economically feasible) can
facilitate inbreeding to homozygosity by selecting progeny at each generation
which are homozygous
over a maximal proportion of the genome.Figure 1
Distribution of percentSolanum lycopersicum genome in the F2  population, estimated based on 220 codominant markers.


## 3.5. Construction of the Linkage Map

A genetic
linkage map was constructed based on 115 RFLP, 94 EST, and 41 RGA loci using the F2 population of 172 individuals. The present map (L  ×  PM3) spanned 1002.4 cM of
tomato genome with an average marker interval length of 4.0 cM (Figure 2). The number of markers per chromosome
ranged from 16 (chrs. 3 and 7) to 28 (chr. 1). Chromosome 1 had the largest
linkage group (102.9 cM) followed by chromosomes 9 and 2 (96.1 and 92.6 cM, resp.),
whereas chromosome 7 had the smallest one (69.8 cM), preceded by chromosomes 4
and 5 (72.2 and 70.6 cM, resp.). Only two regions, on chromosomes 3 and 12,
contained marker intervals larger than 20 cM (Figure 2), and this was mainly
because of the low level of polymorphism between the two parents of this
mapping population for markers on these chromosomes. This map was compared with
several other molecular linkage maps of tomato for marker order, recombination
frequencies, and total map length, as described below.Figure 2
A genetic
linkage map of tomato constructed based on an F2 population of a cross between a tomato (S. lycopersicum)
breeding line (NCEBR-1) and an accession (LA2093) of tomato wild species S. pimpinellifolium and 250 RFLP, EST,
and RGA markers. RFLP markers are shown in blue, ESTs in green, and RGAs in red
fonts. The names of the markers are shown at the right and the map distances
between them (in cM, using Kosambi function) are shown at the left of the
chromosomes. The approximate chromosomal locations of disease-resistance genes
(R-genes) and quantitative resistance loci (QRL), as inferred from other
published researches, are shown in parentheses to the right of chromosomes. The
descriptions of the R-genes and QRL are as follows: Asc: resistance to Alternaria stem canker (Alternaria alternata f. sp. lycopersici)
[54, 55]; Bw (1–5) or Rrs (3–12): QLRs for
resistance to bacterial wilt (Ralstonia
solanacearum) [56–59]; Cf (1–9, ECP2):
resistance to leaf mould (Cladosporium
fulvum) [60–65]; Cmr: cucumber mosaic virus [66]; Fen: sensitivity to herbicide fenthion [67]; Frl: resistance to Fusarium crown and root rot (Fusarium oxysporum f. sp.
radicis-lycopersici) [68]; Hero: resistance to potato cyst namatode (Globodera rostochiensis) [69]; I (I,1, 2, 2C, 3): resistance to different
races of Fusarium wild (Fusarium
oxysporum f. sp. lycopersici) [70–78]; Lv: resistance to powdery mildew (Leveuillula taurica) [79]; Meu-1: resistance to potato aphid [80–82]; Mi (Mi, 1, 2, 3, 9):
resistance to root knot nematodes (Meloidogyne spp.) [81, 83–89]; Ol (1, 2, 3): resistance
to powdery mildew (Oidium lycopersicum)
[90, 91]; Ph (1, 2, 3): resistance
to late blight (Phytophthora infestans)
in tomato [92–94]; Pot-1: resistance to potyvirus [95]; Pto and Prf: resistance
to bacterial speck (Pseudomonase syringae pv tomato) [96, 97]; Py-1: resistance to corky root rot (Pyrenochaeta lycopersici) [98]; Rcm (1–10): QRL for
resistance to bacterial canker (Clavibacter
michiganensis) [99, 100]; Rrs (3–12) or Bw (1–5):
QLRs for resistance to bacterial wilt (Ralstonia
solanacearum) [56–59]; Rx (1, 2, 3, 4):
resistance to bacterial spot (Xanthomonas
campestris) [101–103]; Sm: resistance to Stemphilium [104]; Sw-5: resistance to tomato-spotted wilt virus [105, 106]; Tm-1 and Tm-2a:
resistance to tobacco mosaic virus [68, 107–110]; Ty (1, 2, 3): resistance
to tomato yellow leaf curl virus [111–113]; Ve: resistance to Verticillium dahliae [114, 115].


## 3.6. Mapping of ESTs

The use of the 115 RFLP anchor markers
facilitated mapping of the 94 EST loci onto the 12 tomato chromosomes. The
number of ESTs per chromosome ranged from 4 (chr. 12) to 12 (chr. 10) (Figure2). The use of ESTs as genetic markers has several advantages. First, they can
be used as codominant markers for genetic mapping and QTL identification [53]. Although ESTs were used as RFLP
markers, that is, through Southern hybridization, technically they can be
converted to PCR-based markers adapted to high-throughput analysis. Such
conversion may reduce polymorphism level, in particular between closely related
individuals, though it is expected to enhance their utility as genetic markers.
Second, mapping of ESTs can facilitate association of functionality with
phenotype. EST markers are derived from partial or complete sequences of cDNA
clones, which may provide information on gene function. Third, coding
sequences, especially those of house-keeping genes, are rather conserved across
species. Mapping of ESTs and comparative genomics may lead to the detection of
new genes in different species.Inspections of the distribution of ESTs on different
chromosomes indicated that in some cases they were clustered, for example, ESTs
on chromosomes 4, 8, 10, and 11. Further inspections indicated that chromosomal
locations of some clustered or individual ESTs were colocalized with
approximate locations of some major disease-resistance genes (R-genes) or
quantitative resistance loci (QRLs), as inferred from other published researche (see Figure 2). While such colocalization suggests that these ESTs may be genetically
related to resistance genes or QRL, their actual functionality relationships
can only be determined by further analyses such as isolation and sequencing of
full EST sequences and functional genomic studies.Currently, there are more than 214 000 ESTs identified in
tomato (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=tomato), of which
only a small percentage has been mapped onto the tomato chromosomes (http://www.sgn.cornell.edu/cgi-bin/search/direct_search.pl?search=EST). The ESTs
were derived from more than 23 cDNA libraries [116, 117] and their sequences are
available on Solanaceae Genome Network (SGN; http://www.sgn.cornell.edu/). All but
four (cLET10E15, cLER4F5, cLEC6O2, and cLEG9N2) of the ESTs mapped in the
present study were not previously mapped onto tomato chromosomes. Moreover, of
the four that were previously mapped, different members of the corresponding
contigs were mapped onto the same or different tomato chromosomes as in the
present study. For example, cLET10E15 and cLER4F5 have overlap sequences with
cLET1A5 and cLET3F16, respectively, and were mapped on the same chromosomes (http://www.sgn.cornell.edu/) as in the
present study. cLEC6O2, which was mapped to chromosome 1 in the present study,
was mapped to chromosome 8 and named cLPT1J10 (SGN: F2 population ofa cross S. lycopersicum LA925  ×  S. pennellii LA716). EST
clone cLEG9N2, which was mapped to chromosome 1 in this study, was previously
mapped under cLET20B4 but with no known chromosomal position (http://www.sgn.cornell.edu/). Also, as
indicated earlier, five of the EST clones resulted in two pairs of polymorphic
bands each. For two of these clones, the two polymorphic bands were mapped onto
two linked loci, that is, cTOC2J14a and cTOC2J14b on chromosome 5 and
cLEC14I18a and cLEC14I18b on chromosome 11. Others
were mapped onto different chromosomes; for example, cLEW22D11a was mapped to
chromosome 6 whereas cLEW22D11b to chromosome 4, and cTOE7J7a was mapped to
chromosome 1 whereas cTOE7J7b to chromosome 5.


## 3.7. Mapping of RGAs

PCR
amplification using the 10 pairs of RGA primers (Table2) followed by
denaturing PAGE resulted in the detection of a few hundred polymorphic and
monomorphic bands. As described in Section 2, of the detected bands, 41 were
strong and verifiable and thus were scored in the F2 population. The
amplified fragment size of these RGA bands ranged from 150 to 760 bp. Linkage
analysis indicated that the 41 RGA markers were located on the 12 tomato
chromosomes, ranging in number from 1 (on chrs. 3, 5, and 7) to 9 (on chr. 1)
(Figure 2). The results indicated that RGA loci could be used as genetic
markers for genome mapping, consistent with previous suggestions [28, 30]. In several cases, RGA loci were
clustered, similar to that observed for R-genes
in various plant species [17, 19, 29, 60, 118–120]. For example, on each of
chromosomes 1, 2, 9, 10, 11, and 12, three or more RGA loci that were amplified
from the same or different primer pairs mapped to the same or nearby positions
(Figure 2). This observation indicated that different primers might initiate
amplification of closely linked RGA loci that might be members of the same or
different gene families.Map positions of RGA loci were compared with chromosomal
positions of known tomatoR-genes and major QRL, whose positions were inferred from the previously published maps, as displayed and
described in Figure 2. Most positions were inferred based on linkage to
reference markers and thus should be considered best approximations. Colocalization of RGA loci with R-genes and QRLs were observed on a few
chromosomes, including regions on chromosomes 1, 2, 7, 8, 9, 10, 11, and 12
(Figure 2). These observations suggest the possibility of the presence of R or
DR genes at the locations of RGAs, though this hypothesis could be confirmed
only by extensive mapping and functional analysis of RGAs. Specifically,
mapping of the associated RGAs in populations segregating for the colocalized R-genes
and cloning and molecular characterization of RGAs are necessary before any
functional relationship could be established.The map positions of the RGA loci in the present map (L  ×  PM3) were compared with those reported in aS.
lycopersicum  ×  S.
habrochaites (L  ×  H2) map [28]. There were 19 common RGA loci
between the two populations and 13 (68%) of which mapped to the same locations
in the two maps, suggesting consistent and reproducible positions of RGAs
across populations.


## 3.8. Comparison of the Map with Other Molecular Linkage Maps of Tomato

The
present map (L  ×  PM3) was compared with two previously developedS. lycopersicum  ×  S. pimpinellifolium maps, including L  ×  PM1 [6] and L  ×  PM2 [7] as well as the high-density S. lycopersicum  ×  S. pennellii (L  ×  P) map of tomato [25]. The present map is different
but complementary to L  ×  PM1 and L  ×  PM2 maps in several ways. First, different S.
lycopersicum and S. pimpinellifolium parents
and pretty much different molecular markers were used in the construction of
the three maps. The L  ×  PM1 was constructed based on a cross between a processing
tomato cultivar (M82-1-7) and S.
pimpinellifolium accession LA1589 using ~120 RFLP and RAPD markers. The L  ×  PM2 was constructed based on a cross between a fresh market tomato breeding
line (NC84173) and S. pimpinellifolium accession
LA722 using 151 RFLP markers. The current map (L  ×  PM3) was constructed based
on 250 RFLP, EST, and RGA markers using superior parental lines, as described
earlier. It is expected that this map will have great utilities, including
exploitation of the genetic potential of LA2093 and other S. pimpinellifolium accessions.The second point of
difference is that relatively a small percentage of the markers used in the
present study were used in the previous twoS.
lycopersicum  ×  S.
pimpinellifolium linkage maps. Specifically, a new set of RFLP clones that
detect polymorphism between S.
lycopersicum and S. pimpinellifolium has
been identified in the present study, beyond those that were identified in the
construction of the previous two maps. However, an important observation is
that markers that are polymorphic in one L  ×  PM cross usually have a greater
chance of being polymorphic in other L  ×  PM crosses, compared to markers
directly chosen from the high-density L  ×  P map. Nonetheless, the observation
that only 54% of the mapped RFLP clones in the L  ×  PM2 population were
polymorphic in the L  ×  PM3 population indicates the presence of considerable
DNA sequence variation among S.
pimpinellifolium accessions. The overall results suggest that while for
each S. pimpinellifolium accession
new polymorphic markers need to be identified, the most useful sources would be
those markers that have already been mapped in other S. lycopersicum  ×  S.
pimpinellifolium crosses. Third, unlike in
the previous two L  ×  PM maps, in the present map, “functional” markers such as
ESTs and RGAs were used. Such markers may be more useful than random genetic
markers for identification of candidate genes. The use of a large number of
markers and the incorporation of functional markers in the present map extends
its practical value in various genetics and breeding studies. However, the
availability of three L  ×  PM maps with rather different molecular markers should
facilitate marker-assisted exploitation of these and other S. pimpinellifolium accessions.When the current map was compared with L  ×  PM1 [6], L  ×  PM2 [7], and the high-density L  ×  P map [25], it was determined that the
linear order of
the common markers were generally the same. However, there were differences in
interval lengths for several adjacent markers. For example, of 13 common marker
intervals between L  ×  PM3 and L  ×  PM2 maps, 6 intervals on chromosomes 2, 3, 5,
6, and 12 differed in length by 2-3 fold, of which 1
interval was expanded in L  ×  PM3 map. The difference between the two maps in
marker interval lengths was not unexpected given the use of different type populations (F2 versus BC1), rather small
size populations (172 and 119) and different number of markers (250 versus
151), all of which could have affected the occurrence and detection of
recombination in different intervals. When the L  ×  PM3 was compared with the
high-density L  ×  P map, which was constructed based on >1 000 genetic
markers and 67 F2 plants, genetic distances differed markedly for a
large number of marker intervals. For example, for 36 common marker intervals,
genetic distances differed between the two maps by at least twofold; of these,
7 intervals (23%) showed decreased and 13 (36%) showed increased recombination
in the L  ×  PM3 map. Greater differences in marker interval lengths between L  ×  PM3 and L  ×  P maps compared to that between L  ×  PM3 and L  ×  PM2 maps was not unusual
considering the relatively close phylogenetic relationships between the L  ×  PM3
and L  ×  PM2 mapping populations.When comparing the L  ×  PM3 map with the high-density L  ×  P
map, the most striking differences in genetic distances were observed in
centromeric regions of chromosomes 3, 4, and 9, where substantial expansions in
map distances were observed in the L  ×  PM3 map, and in two locations on
chromosome 12, where substantial contractions were observed in the L  ×  PM3 map
(Table4 and Figure 1). The
decrease in recombination frequencies in the centromeric regions of tomato
chromosomes was previously attributed to the centromeric suppression of
recombination [5, 121, 122]. Such suppression was suggested
to be more frequent in wider crosses than in intraspecific crosses and crosses
between closely related species. Further inspections indicated that the
differences in genetic distances between the two maps across the rest of the
genome were generally interval specific and not a characteristic of individual
chromosomes. For example, for chromosomes 2, 3, 6, 10, 11, and 12, the L  ×  PM3
map exhibited expansion in some intervals and contraction in others (Table 4).
As indicated earlier, such differences were due in part to the detection of
chance recombination given the limited population sizes used in these studies.Table 4
Comparison of map distances based on common marker
intervals between three molecular linkage maps of tomatoa.
IntervalChr.Marker interval map distance (cM)(L  ×  PM3)b(L  ×  PM2)c(L  ×  PM3)/(L  ×  PM2)(L  ×  P)d(L  ×  PM3)/(L  ×  P)TG70-TG273124.323.81.08.92.7*TG554-TG45326.6——0.0NA*TG453-TG14520.4——6.80.1*TG145-CT103210.3——10.11.0CT176-TG58225.618.90.3*15.50.4*CT59-TG62022.67.00.4*0.0NA*TG114-TG13230.08.9N/A*15.4NA*TG66-CT225B36.6——1.83.7*CT225B-CT8239.8——6.31.6CT82-TG515312.714.80.93.93.3*TG123-TG182412.5——14.20.9TG182-TG60946.7——5.51.2TG609-CT178411.8——11.11.1CT167-CT93519.38.92.2*12.91.5TG503-TG9652.5——3.20.8TG274-TG59064.6——10.40.4*TG356-TG365613.011.91.14.13.2*C54-TG27964.010.20.4*—NATG183-TG128715.7——2.36.8*TG128-CT22673.13.50.91.61.9TG128-TG174719.313.61.410.71.8TG176-CD4088.7——0.0NA*CT265-TG294812.89.61.313.11.0TG486-CD391.7——1.31.3CD3-CT279911.3——5.62.0*CT279-TG3591.9——0.0NATG408-CD34104.9——22.90.2*CD34-TG4031030.137.50.87.44.1*TG629-TG497110.0——0.0NATG30-CT651116.7——3.94.3*TG68-CT79123.36.70.5*14.40.2*CT99-TG618125.4——0.86.8*TG618-TG1111212.5——6.12.0*TG111-TG565123.0——0.0NA*CT156-TG473126.1——19.10.3*TG473-CD2120.0——1.80.0aOnly common
marker intervals that were different in length by at least twofold between L  ×  PM1 and either
L  ×  PM2 or L  ×  P linkage maps are shown.bL  ×  PM3: Solanum lycopersicum (NCEBR-1)  ×  S. pimpinellifolium (LA2093) map
(present map).cL  ×  PM2: S. lycopersicum (NC84173)  ×  S. pimpinellifolium (LA722) map
[7]
.dL  ×  P: S.lycopersicum (VF36-Tm2)  ×  S. pennellii (LA716) map
[25]
.*Difference
in interval length by at least twofold. Dashes (—) indicate
no common interval for comparison. NA indicates a number divided by 0.0, 0.0
over a number, or no comparison was made.Comparisons were also made across the four maps (L  ×  PM3, L  ×  PM2, L  ×  PM1, and L  ×  P) in terms of individual chromosome and total map
lengths. The total length of the current map (1002 cM) was comparable with that
of the L  ×  P (1277 cM), L  ×  PM1 (1275 cM), and the L  ×  PM2 (1186 cM) maps.
Furthermore, across the maps the length of each chromosome in the current map  was comparable to the corresponding chromosome in the other
maps (Table5).Table 5
Pairwise comparison of the present map (L × PM3) with other maps of tomato for individual chromosome lengths based on orthologous markers.
Chromosome length (cM)Linkage mapa123456789101112AverageTotalL × PM3102.992.685.372.270.674.669.886.696.180.688.383.483.61003.0L × PM2129.7121.9133.810894.182.891.364.4104.884.978.292.698.91186.5L × PM1149.698.2116.697.2108.285.2116.486.1104.2101.5107105.2106.31275.4L × P133.5124.2126.1124.897.4101.991.694.911190.18893.1106.41276.6L × PM3/L × PM20.80.80.60.70.80.90.81.30.90.91.10.90.8L × PM3/L × PM10.70.90.70.70.70.90.61.00.90.80.80.80.8L × PM3/L × P0.80.70.70.60.70.70.80.90.90.91.00.90.8*L × PM3, S. lycopersicum (NCEBR-1) × S. pimpinellifolium (PSLP125) map (the present map); L × PM2, S. lycopersicum (NC84173) × S. pimpinellifolium (LA722) map [7]; L × PM1, S. lycopersicum (M82-1-7) × S. pimpinellifolium (LA1589) map [6]; E × P, S. lycopersicum (VF36-Tm2) × S. pennellii (LA716) map [25].


## 4. Conclusion

A
medium-density molecular linkage map of tomato is developed based on a cross
betweenS. lycopersicum and S. pimpinellifolium, two
phylogenetically closely related species. The parents of this map are superior
genotypes and are expected to be useful for tomato crop improvement. This map
will provide a basis for the identification, characterization, and
introgression of useful genes and QTLs present in LA2093 and other S. pimpinellifolium accessions. It will
also facilitate studies of gene and genome organization and evolution,
dissection of complex traits, and targeted gene cloning. The map includes
different types of molecular markers and provides a basis for identifying and
adding other markers. The genomic locations of several EST and RGA markers
coincided with locations of several known tomato
R-genes or QRL, suggesting that candidate gene approach may be an
effective means of identifying and mapping new R-genes and defining the genetic content of specific chromosomal
regions. Because of the close phylogenetic relationship between the two species
and the past frequent introgression of DNA from S. pimpinellifolium into S.
lycopersicum, this map is expected to be particularly useful to breeding
programs that exploit intraspecific variability within the cultivated tomato.
The combined information from this and the two previously published S. lycopersicum  ×  S. pimpinellifolium maps will facilitate
further identification and exploitation of genetic variation within S. pimpinellifolium, S. lycopersicum var. cerasiforme, and S.
lycopersicum.



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*Source: 926090-2009-02-11.xml*