the 
 tomato solanum 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 . 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 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 . 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 . 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 , 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 . 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 . in addition , during the past 
 several decades , extensive genetic introgressions from this species into the 
 cultivated tomato have been made through plant breeding [ 810 ] . 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 [ 1113 ] , 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 [ 1721 ] . although not all amplified 
 products may correspond to functional disease resistance genes , 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 . 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 of s. 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   ( referred to as l      pm1 map ) and chen and foolad   ( 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 . 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 total of 900 
 f2 individuals were grown under field conditions and screened for 
 various characteristics . among other traits , 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 , 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 . 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 . agarose gel electrophoresis , 
 southern blotting , hybridizations , and autoradiography were conducted as 
 described elsewhere . probes were labeled with [ p]dctp 
 by primer extension . 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 . 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 , the polymorphic ests were 
 used as rflp probes to genotype the f2 individuals , examine their 
 segregation , and map onto the tomato chromosomes . ten pairs of 
 oligonucleotide primers , previously designed based on conserved lrr , nbs , and 
 ptokin motifs of several resistance genes , were used ( table 2 ; ) . 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 ] . briefly , each amplification was performed in a 25-l 
 volume consisting of 300 m 
 each of datp , dctp , dgtp , and dttp , 5 mm of mgcl2 , 1 unit of taq dna polymerase , 2.5 l of 10x buffer ( pcr core system i ; promega , 
 madison , wis , usa ) , 2 l 
 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 94c for an initial denaturation , and 36 cycles of 
 1 minute at 94c ( dna denaturation ) , 1 minute at 50c 
 ( primer annealing ) , and 1.5 minutes at 72c ( primer extension ) , 
 followed by a final 7-minute extension at 72c . denaturing polyacrylamide gel 
 electrophoresis ( page ) was used to separate and detect individual rga bands . briefly , a denaturing gel ( 7 m urea6% 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 50c . twelve l 
 of loading buffer ( 10 m urea0.08% xylene cyanole ) were added to each 25 l 
 amplified dna sample and the mixture was denatured at 95c 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 . each gel accommodated 60 dna samples 
 and three dna size markers ( 1 kb , 100 bp , 50 bp ) . 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 . 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 p - 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 . page polymorphic and monomorphic 
 fragments were excised from the dried polyacrylamide gel and reamplified , by 
 using a needle scratching and pcr reamplification method . 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 . 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 
 (  ) 
 goodness - of - fit analysis . multipoint linkage 
 analysis of the genetic markers in the f2 population was performed 
 using the mapmaker program v. 3.0   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   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 . rflp clones 
 were chosen from two sources , a previously published s. lycopersicum ( nc84173 )      s. pimpinellifolium ( la722 ) linkage map 
 ( l      pm2 )   and the high - density s. lycopersicum ( vf36 -tm2 )      s. 
 pennellii ( la716 ) linkage map of tomato ( l      p ) . 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   and chen and foolad   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 . in the present study , a total 
 of 117 polymorphic rflp clones were used to construct the backbone linkage map . 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 . 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 . 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 ) . 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   or the previous s. lycopersicum      s. pimpinellifolium maps [ 6 , 7 ] , where no such selections were 
 practiced . 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 , 4045 ] . a survey of recently published 
 results of interspecific crosses of tomato indicated that skewed segregation 
 was 8.3% in the l      pm1 bc1 population , 9.9% in the l      pm2 bc1 population , 51% in a s. lycopersicum      s. 
 cheesmaniae ( l      ch ) f2 population , 69% in a s. lycopersicum      s. 
 chmielewskii ( l      cl ) bc1 population , 15% in a s. lycopersicum      s. 
 habrochaites ( l      h1 ) bc1 population , 62% in the l      h2 bc1 population , and 
 80% in a s. lycopersicum      s. 
 pennellii ( l      p ) f2 population . 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   and the low level of skewed 
 segregation in the l      h1 bc1 populations   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 , 5052 ] . 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 24 backcrosses , 
 far more rapid than the 46 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 . 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 . 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 . the use of the 115 rflp anchor markers 
 facilitated mapping of the 94 est loci onto the 12 tomato chromosomes . first , they can 
 be used as codominant markers for genetic mapping and qtl identification . 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 . 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 . pcr 
 amplification using the 10 pairs of rga primers ( table 2 ) 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 . 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 , 118120 ] . 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 tomato r - 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 a s. 
 lycopersicum        s. 
 habrochaites ( l      h2 ) map . 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 . the 
 present map ( l      pm3 ) was compared with two previously developed s. lycopersicum      s. pimpinellifolium maps , including l      pm1   and l      pm2   as well as the high - density s. lycopersicum      s. pennellii ( l      p ) map of tomato . 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 two s. 
 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 , l      pm2 , and the high - density l      p map , 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 
 ( table 4 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 . 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 furthermore , across the maps the length of each chromosome in the current map   was comparable to the corresponding chromosome in the other 
 maps ( table 5 ) . a 
 medium - density molecular linkage map of tomato is developed based on a cross 
 between s. 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 .
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 an f2   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 .