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Estimating genome conservation between crop and model legume species

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Estimating genome conservation between crop and model legume species
  Estimating genome conservation between crop andmodel legume species Hong-Kyu Choi †‡ , Jeong-Hwan Mun †‡ , Dong-Jin Kim †‡ , Hongyan Zhu † , Jong-Min Baek § , Joanne Mudge ¶ , Bruce Roe  ,Noel Ellis †† , Jeff Doyle ‡‡ , Gyorgy B. Kiss †§§ , Nevin D. Young ¶ , and Douglas R. Cook †§¶¶ † Department of Plant Pathology and  § College of Agricultural and Environmental Sciences Genomics Facility, University of California, One Shields Avenue,Davis, CA 95616;  ¶ Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108;   Advanced Center for Genome Technology, Universityof Oklahoma, Norman, OK 73019;  †† John Innes Centre, Norwich NR4 7UH, United Kingdom;  ‡‡ Department of Plant Biology, Cornell University,Ithaca, NY 14853; and  §§ Biological Research Center, Institute of Genetics, H-6701 Szeged, HungaryEdited by Susan R. Wessler, University of Georgia, Athens, GA, and approved August 13, 2004 (received for review March 30, 2004) Legumes are simultaneously one of the largest families of cropplants and a cornerstone in the biological nitrogen cycle. Wecombined molecular and phylogenetic analyses to evaluate ge-nomeconservationbothwithinandbetweenthetwomajorcladesof crop legumes. Genetic mapping of orthologous genes identifiesbroad conservation of genome macrostructure, especially withinthe galegoid legumes, while also highlighting inferred chromo-somal rearrangements that may underlie the variation in chromo-some number between these species. As a complement to com-parative genetic mapping, we compared sequenced regions of themodellegume Medicagotruncatula withthoseofthediploid Lotus japonicus  and the polyploid  Glycine max  . High conservation wasobserved between the genomes of  M. truncatula  and  L. japonicus ,whereas lower levels of conservation were evident between  M.truncatula  and  G. max  . In all cases, conserved genome microstruc-turewaspunctuatedbysignificantstructuraldivergence,includingfrequentinsertion  deletionofindividualgenesorgroupsofgenesandlineage-specificexpansion  contractionofgenefamilies.Theseresults suggest that comparative mapping may have considerableutility for basic and applied research in the legumes, although itspredictive value is likely to be tempered by phylogenetic distanceand genome duplication. T he Fabaceae, or legumes, are cultivated on 180 millionhectares, involving   12% of Earth’s arable land and ac-counting for  27% of the world’s primary crop production (1).Their unusual capacity for symbiotic nitrogen fixation underliestheir importance as a source of protein in the human diet and of nitrogen in both natural and agricultural ecosystems. Legumesare also increasingly recognized as a source of valuable second-ary metabolites. These factors have fueled a significant increasein legume research over the past decade.The   20,000 legume species are divided into three subfami-lies: Mimosoideae, Caesalpinioideae, and the numerically andeconomically dominant Papilionoideae (2). With the notableexception of peanut, the important crop legumes occur in twoPapilionoid clades, referred to here as the ‘‘phaseoloid’’ and‘‘galegoid’’ legumes (Table 1). Despite their close phylogeneticaffiliations (Fig. 1), the genetic systems represented within thisgroup are diverse, ranging from simple autogamous diploids tocomplex out-crossing polyploids. Genome size also varies widelyamong legumes, with pea having a genome size 10 times that of some related diploid genera.The large number of important legume species precludes theirsimultaneous in-depth characterization. Moreover, several croplegumes have one or more characters (e.g., medium to largegenomes and  or polyploid nature) that limit their utility asexperimental systems. Two legumes with favorable genetic at-tributes, namely  Medicago truncatula  and  Lotus japonicus , havebeen selected as model species and are the focus of largemultinational genome projects. The early fruits of working withthese well characterized genomes are evident in the recentadvances in our understanding of symbiotic nitrogen fixation inboth  M. truncatula  and  L. japonicus  (3). A pressing need in legume genomics is to integrate knowledgegained from the study of model legume genomes with thebiological and agronomic questions of importance in the cropspecies. Comparative genetic mapping is well established inseveralplantfamilies,mostnotablythePoaceae(4),whereinitialstudies predicted that synteny would greatly facilitate genediscovery among related species (5, 6). However, even closelyrelated grass species (7, 8), in some cases members of the samespecies (9), can exhibit significant divergence in genome orga-nization. It is important to know whether similar features areprevalent in other plant families, in particular because the extentof such differences may define the limits of comparative struc-tural genomics as a strategy for applied agriculture.Here we combined genetic and phylogenetic analyses to mapputatively orthologous genes across seven legume species. Com-plementing the genetic linkage analysis, we surveyed the con-servation of genome microstructure between  M. truncatula  and  L. japonicus  and  M. truncatula  and  Glycine max  (soybean) bycomparing fully sequenced bacterial artificial chromosome(BAC)clones.Thecombinedgenetic,phylogenetic,andgenomicanalyses demonstrate extensive conservation of gene order andorthology between the crop and model legumes and also identifyfeatures of structural divergence between these genomes. Materials and Methods Plant Material and Mapping Populations.  Plant genotypes used forgenetic mapping are shown in Table 1. The core maps for eachofthegenomesunderanalysishavebeendescribed(6–15).Inthecases of   Pisum sativum  and  Vigna radiata , mapping populations were composed of highly selected genotypes chosen so thatrecombination break points were spaced evenly throughout thegenome (16). BAC clones were mapped in  M. truncatula  bymeans of simple sequence repeat polymorphisms discovered inthe course of shotgun sequencing. Development of Cross-Species Genetic Markers.  The developmentand genetic mapping of gene-specific markers were as describedby Choi  et al.  (10).  BLASTN  [National Center for BiotechnologyInformation (NCBI), Bethesda] was used to identify conservedsequences between ESTs of the legume  M. truncatula  and otherlegume species. Multiple sequence alignments, with the  Arabi- dopsis  genomic sequence used to infer intron position, facilitateddesign of PCR primers that anneal to conserved exon sequencesand amplify across more diverged introns. Polymorphisms (Ta-ble 2, which is published as supporting information on the PNAS web site) were identified by sequencing PCR products from This paper was submitted directly (Track II) to the PNAS office.Abbreviations: BAC, bacterial artificial chromosome; NCBI, National Center for Biotech-nology Information. ‡ H.-K.C., J.-H.M., and D.-J.K. contributed equally to this work. ¶¶ To whom correspondence should be addressed. E-mail:© 2004 by The National Academy of Sciences of the USA  cgi  doi  10.1073  pnas.0402251101 PNAS    October 26, 2004    vol. 101    no. 43    15289–15294       A      G      R      I      C      U      L      T      U      R      A      L      S      C      I      E      N      C      E      S  parental lines (Table 1), followed by manual inspection of alignmentsandchromatograms.Markersweretypicallyanalyzedas cleavable amplified polymorphic sequences (10). Single-nucleotide polymorphisms that did not alter a restriction site were scored by DNA sequencing of PCR products. Resequenc-ing was used to confirm or refute apparently ambiguous data. Phylogenetic Analysis.  Neighbor-joining trees were rooted byusing the closest  Arabidopsis  sequence as an outgroup or leftunrooted where no close homolog was present in  Arabidopsis .Phylogenetic analyses were conducted by using parsimony op-tions in  PAUP * (17). The principal analysis involved 100 searches with random taxon addition and tree bisection–reconnection(TBR) branch swapping, with maxtrees set to increase withoutlimit. Support for branches was assayed by 100 bootstrap repli-cate searches using simple taxon addition, TBR branch swap-ping, and maxtrees set to 1,000. Microsynteny Analysis.  Accession numbers for sequenced BACsare given in Tables 2–5, which are published as supportinginformationonthePNASwebsite.Homologoustransformation-competent BAC (TAC) clones of   L. japonicus  were obtainedfrom NCBI (18).  Ab initio  gene prediction involved the eudicot version of   FGENESH  (  berry.phtml?topic   gfind). Gene prediction based on identity to transcribed se-quences was obtained by  BLASTN  against The Institute forGenomic Research  M. truncatula  or  L. japonicus  Gene Index databases (  tdb  tgi). Additional predicted proteins were identified by means of   BLASTX  (NCBI) against the NCBInonredundant protein database.  BLASTP  (NCBI) was used tocompare predicted proteins between  M. truncatula  and  L. ja- ponicus  clone pairs, with a maximum  E  value cutoff of   e  10 andamedian  E  valueof    e  100 for533proteinpairs. REPEATMASKER (http:   cgi-bin  Repeat-Masker) was used to screen interspersed repeats, transfer RNA,and low-complexity DNA sequences. Results Development of Cross-Species Gene-Specific Genetic Markers.  Wesought to develop cross-species genetic markers where locusorthology was an explicit aspect of the analysis. One hundredsixty-seven gene-specific PCR primer pairs were tested foramplification and polymorphism by using the parents of avail-able mapping populations in  M. truncatula ,  Medicago sativa ,  Pi. sativum ,  L. japonicus ,  V. radiata ,  Phaseolus vulgaris , and  G. max (Table 1). To test the orthology of these genetic markers, weconstructed phylogenies for 24 of the markers that producedhigh-quality sequence data for at least four of the six speciesunder analysis (excluding  M. sativa ) (as shown by example in Fig.6, which is published as supporting information on the PNAS web site). Combining alignment matrices and analyzing the databy neighbor-joining and maximum parsimony methods yielded atree depicting genomic relationships. The monophyly of thegaleoid and phaseoloid clades was strongly supported, as was thesister relationship of   Phaseolus  and  Vigna . Analysis of individualgene trees supports the Loteae as sister to the galegoid clade. Asa further test of sequence orthology, 11 markers with unambig-uous global alignments were analyzed across 95 diploid legumespecies (Fig. 7, which is published as supporting information onthe PNAS web site) spanning the diversity of the Fabaceae. Theoverall agreement with published phylogenies of the family (2,19) supports the orthology of these genes and the utility of theseexon-derived markers as tools for comparing legume genomes. Macrosynteny Analysis Among  M. truncatula  ,  M. sativa  ,  Pi. sativum  , G. max  ,  V  .  radiata  , and  Ph. vulgaris  .  For purposes of establishing acomparative genetic map spanning galegoid and phaseoloidspecies, we analyzed marker segregation in  M. truncatula ,  M. sativa ,  Pi. sativum , and  V. radiata . In addition to the markersdeveloped based on phylogenetic criteria, we analyzed 60 primerpairs developed based on homology to genetic markers in G.max and 117 additional markers developed for the  M. truncatula  coregenetic map (10). In all cases,  M. truncatula  was the central pointof comparison. Comparisons between the two  Medicago  speciesand between  Ph. vulgaris  and  V. radiata  have been presentedelsewhere (11, 12) and are included here for the sake of integration.The pea genome is much larger (  10 times) than that of   M.truncatula  and has a base chromosome number of 7, comparedto 8 in  M. truncatula . Despite these overt differences, analysis of 57 gene-specific markers reveals broad conservation of genomestructure, with the major evident differences being sites of  Table 1. Attributes of species used for synteny analysis Species Common nameGenome size,Mbp  N   Tribe Clade SL PL Genotypes M. truncatula  Barrel medic 500 8 Trifoleae Galegoid 183 130 A17, A20, DZA M. sativa  Alfalfa 1,600 16 Trifoleae Galegoid 70 68 Mscw2, Msq93 Pi. sativum  Pea 5,000 7 Viceae Galegoid 101 68 JI15, JI281, JI399, JI194 G. max   Soybean 1,100 20 Phaseoleae Phaseoloid 56 15 PI209322, Evans V. radiata  Mung bean 520 11 Phaseoleae Phaseoloid 62 31 TC1966, VC3890 Ph. vulgaris  Common bean 620 11 Phaseoleae Phaseoloid 37 22 BAT93, Jalo L. japonicus  Bird’s foot trefoil 500 6 Loteae 67 44  Lotus filicaulis, L. japonicus Gifu SL, sequenced loci; PL, polymorphic loci;  N  , gametic chromosome number. Fig. 1.  Taxonomic relationships within the two major clades of crop le-gumes, the prevailing view of phylogeny for the species under analysis, withdivergence times estimated based on Penalised Likelihood analysis (2). Mostcrop legumes occur either within the galegoid clade, including tribes Viceae,Trifolieae,andCicereae,orwithinthephaseoloidclade,whichissynonymouswith the tribe Phaseoleae. MYA, million years ago. 15290    cgi  doi  10.1073  pnas.0402251101 Choi  et al  .  inferred chromosomal rearrangements. An average of eightcolineargeneticmarkerswerepresentforeachpeachromosome,and in only one case (i.e., marker PTSB) did we identify thepossible translocation of an orthologous gene. Instead, weidentifiedalimitednumberofchromosomaltranslocationeventssuch as that shown for the top terminal region of PsLGIII andthe bottom portion of MtLG2 (Fig. 2). MtLG6 could not beeffectively integrated into the  Pi. sativum  genetic map, due to alack of comparative markers in this linkage group. This resultcorresponds with the previous observation that MtLG6 is rich inheterochromatic DNA (20) and relatively poor in transcribedgenes (10). The absence of a corresponding single linkage groupin pea suggests that chromosomal rearrangements involving  M.truncatula  chromosome 6 might be responsible for the differencein chromosome number between these two species. V. radiata  (mung bean) and  Ph. vulgaris  (common bean)represent closely related members of the  Phaseoleae , both witha chromosome number of each other. The genetic analysis of 22gene-specific markers and 16 PCR markers converted fromrestriction fragment length polymorphisms (11, 12) revealed acombination of marker colinearity, inferred translocations orduplications, and nonsyntenic loci (Figs. 2 and 8, which ispublished as supporting information on the PNAS web site).Twenty-nine of the 38 markers tested revealed evidence of conserved gene order between  M. truncatula  and  V. radiata . Theremaining nonsyntenic markers either mapped distal to regionsof colinearity or their positions interrupted syntenic markers. Inmany cases, syntenic genes bracketed inferred rearrangements,such as the possible duplication  translocation of markers pA257and pA315 on  Vigna  LG8  9 (Fig. 2) and the putative single genetranslocation of markers ARG10 and PPH on  Pi. sativum  LGVIto  Vigna  LG11 and LG1, respectively (Fig. 2). In the mostextreme case,  Vigna  LG1 (Fig. 8  F  ) contains seven markers thatmap to four different  M. truncatula  linkage groups, with twopossible cases of conserved synteny (i.e., the syntenic markersPNDKN1, SUSY, and CPOX2 map to  M. truncatula  LG8, whereas the syntenic markers MMK1 and pA487 map to  M.truncatula  LG4). In contrast, broad synteny was observed amongMtLG1-PsLGII-VrLG4, indicating that this linkage group mightbe ancestral to the galegoid and phaseoloid legumes (Fig. 8  A ).Soybean is also a member of the phaseoloid clade but has apolyploid genome that is predicted to have undergone duplica-tion since its divergence from other  Phaseoleae . Sixty loci weremappedincommonbetween  M.truncatula andsoybean,withthemajority of markers derived from homologs of soybean restric-tion fragment length polymorphism probes identified in  M.truncatula  (10). A complicating feature of cultivated soybean isalowlevelofpolymorphism,asevidencedinTable1,whichwhentaken together with the high level of soybean gene duplicationsignificantly reduced our ability to interpret the comparativemap between these two species. Nevertheless, 38% of themarkers revealed putative synteny between  M. truncatula  and Fig. 2.  Macrosyntenic relationships among legumes with reference to  M. truncatula  linkage groups 2 and 3. Mt,  M. truncatula ; Ms,  M. sativa ; Ps,  Pi. sativum ;Vr,  V. radiata ; Gm,  G. max  ; Pv,  Ph. vulgaris . Shared comparative markers are denoted by bold lettering. Extrapolated marker positions are denoted by dottedsemicircle lines and are drawn only when the colinearity is conserved in the neighboring regions. The comparison between  V. radiata  and  Ph. vulgaris  is basedon previous work (11–15). Choi  et al  . PNAS    October 26, 2004    vol. 101    no. 43    15291       A      G      R      I      C      U      L      T      U      R      A      L      S      C      I      E      N      C      E      S  soybean,identifying11colinearblocksbetweenthetwogenomes(Figs. 2 and 8). Yan  et al.  (21, 22) report genome-wide conservedmicrosynteny between the genomes of   M. truncatula  and soy-bean, with 54% of 50 soybean contig groups showing conservedmicrosynteny to  M. truncatula . Five of the extensively microsyn-tenic contigs (21) were mapped in  M. truncatula  in this study,three of which, namely  A095 ,  A064 , and  A315 , were mapped inregions showing putative synteny between  M. truncatula  andsoybean. Microsynteny Among Papilionoid Legume Genomes.  To determinethe extent to which macrosytenic relationships identified bygenetic mapping are indicative of conserved genome microstruc-ture,wecomparedputativelyorthologouslargeinsertclonepairs[i.e., BAC or transformation-competent BAC (TAC) clones]between  M. truncatula  and  L. japonicus  and between  M. trun- catula  and soybean. The Loteae are a sister group to the galegoidlegumes (Fig. 1), and, thus,  L. japonicus  has a more recentancestry to  M. truncatula  than to soybean. Sixty-three sequencedBAC and TAC clone pairs containing an average of ninemicrosyntenic gene pairs were mapped between the  M. trunca-tula  and  L. japonicus  genomes. As shown in Fig. 3, the genomesare highly syntenic, with macrosynteny punctuated by rearrange-ments that frequently involve translocation of chromosome arms(Fig. 3), reflecting the difference of six chromosomes in  L. japonicus  vs. eight chromosomes in  M. truncatula .Ten clone pairs with broadly spaced genetic positions in thetwo genomes were selected for comparison of microsynteny.Gene content was predicted by a combination of   BLASTN  againstlegume EST databases and  ab initio  prediction by using the dicot version of   FGENESH .  BLASTP  was used for comparison amongspecies. Counting tandem duplications as single homologs andexcluding mobile DNAs, 91 and 84 genes were identified in  M.truncatula  and  L. japonicus , respectively, with 72 (82%) con-served homologs (see Table 4 for a complete list of predictedgenes). With four exceptions, all homologs were present inconserved order and transcriptional orientation. Tandem dupli-cation increased the number of predicted genes in  L. japonicus and  M. truncatula  by 12% and 17%, respectively, with only oneexample of the same homolog duplicated in both species.Moreover, of 18 transposon sequences identified in  L. japonicus and 8 identified in  M. truncatula , only a single example of asyntenic transposon was observed.The example of a 141-kb region of   M. truncatula  at geneticmarker  MtEIL  is shown in Fig. 4. All 16 predicted  M. truncatula genes possess strong similarity to annotated genes in the  Arabi- dopsis  genome. A remarkable feature of this region is thefrequent occurrence of local gene duplication, including twoargonaut-like genes (  MtEIL-e1–2 ), two blue-copper-bindingproteins (  MtEIL-k1–2 ), five kinase-like genes (  MtEIL-l1–4 ), andtwo I-box-binding factors (  MtEIL-m1–2 ). Analysis of the corre-sponding 97-kb segment from  L. japonicus  (  LjEIL ) revealedregion-wide colinearity with the  MtEIL  contig. Ten distinctgenes were identified in  LjEIL , with only a single case of tandemduplication (  LjEIL-d1–2 ). All 10  L. japonicus  genes and atransfer RNA  Leu had homologs in the  MtEIL  region. Six of the16 distinct homologs from the  MtEIL  and  LjEIL  regions exhibita network of microsynteny with two homeologous regions of   Arabidopsis  chromosomes 2 and 5, respectively (Fig. 4).Similar analyses were conducted for two BAC clones at the  rgh1  locus of soybean and putatively orthologous BAC clonesfrom  M. truncatula . In total, 22 genes were identified in  M.truncatula and21genesinsoybean(Table5).Fourteenhomologs(63%) were conserved between the two genomes, all in the sameorder and transcriptional orientation. Two cases of tandemduplication were observed, one in each species, and no trans-poson sequences were identified within the syntenic region of either species. With one exception, all of the predicted genespossessed homology to predicted or known proteins in  Arabi- dopsis ,suggestingthatthegenesabsentfromthesyntenicregionsof   M. truncatula  or soybean are likely to be present elsewhere in Fig. 3.  Macrosyntenic relationship of  M. truncatula  and  L. japonicus . Sixty-three pairs of sequenced BAC clones (Table 3), representing putatively or-thologous loci with known genetic map positions, were used to construct acomparative map between  M. truncatula  and  L. japonicus . Based on theindependentselectionofclonesineachspecies,manyclonepairspossessonlypartial overlap. Line color indicates the number of conserved genes betweentwo clones: black, two; blue, three to four; red, five or more. Fig. 4.  Microsynteny between  M. truncatula  and  L. japonicus . Microsyntenyat the MtLG5 locus MtEIL2–1. Gene annotations are given in Table 4. Genesshown in pink correspond to genetic markers. Letters denote unique geneannotations, with numbers denoting tandem duplication. 15292    cgi  doi  10.1073  pnas.0402251101 Choi  et al  .  their respective genomes. This single colinear region of thelegume genomes shares abbreviated stretches of microsynteny with three separate regions of the  Arabidopsis  genome, as shownin Fig. 9, which is published as supporting information on thePNAS web site. Discussion The idea that conserved genome synteny can facilitate transferof knowledge among related species of plants is best articulatedin the case of the grasses (5, 6). It is increasingly clear, however,that there are many exceptions and complexities to the ‘‘rule’’ of conserved grass genome synteny, because even in regions of genetically defined synteny the insertion  deletion and duplica-tion of genes can contribute to significant divergence (e.g., refs.7 and 8). Recent studies using the genome of   Arabidopsis  as areference document a history of genome duplication in angio-sperms (23–25), followed by significant erosion of local genecontent. In retrospect, it appears that genome synteny is unlikelytobewellconservedbeyondthetaxonomiclevelofplantfamilies(4). As a consequence, testing the ‘‘grass model’’ of genomesynteny for other agronomically important families is an impor-tant objective for plant sciences.In the case of the legume family of plants, there are numerousspecies with a long history of traditional breeding but withlimited molecular characterization, and there are several speciesthat are characterized at both the genetic and genomic levels.Determiningtheextentofsynteny(andthefrequencyandnatureof its exceptions) among these legume genomes was the focus of this study. We report that synteny is high among closely relatedspecies, and that the degree of synteny declines with increasingphylogenetic distance. Although this study is unusual in its useof explicit phylogenetic measures to assess gene orthology and itsincorporation of a large genome sequence data set, the featuresof genome conservation and divergence that we describe aretypical of those observed in comparative analysis of both plantand animal genomes.The high level of conservation between the genomes of   M.truncatula  and  Pi. sativum  is particularly striking given the 10times larger genome (26) and one less chromosome in  Pi. sativum . Much of the expansion in  Pi. sativum  genome size maybe due to retroelements (27), but, whatever the mechanism, ithas done little to disrupt macrosyntenic relationships.  M. trun- catula  LG6 could not be effectively integrated into the  Pi. sativum geneticmap.MtLG6isinterestingforseveralreasons:( i )it is the shortest and most highly heterochromatic of the chro-mosomes (20), ( ii ) it is underrepresented for randomly selectedEST markers (5), and ( iii ) it is remarkably rich in resistance geneanalogs (RGAs) (28). The inability to establish synteny in thisstudy between MtLG6 and  Pi. sativum  is undoubtedly due to thelow frequency of non-RGA EST markers in MtLG6 (10) and thefact that the genetic maps of pea (13) are not well populated byRGA markers. However, parallel analyses conducted by Kalo´  et al. (29)suggestthat  M. sativa LG6issyntenicwithseveralregionsin the pea genome, in particular PsLGVI and PsLGVII (Fig. 7  d ).The absence of a corresponding single linkage group in peasuggests that chromosomal fission  fusion events involving  Medi- cago  chromosome 6 might be responsible for the reduction of chromosome number in  Pi. sativum .  L. japonicus  (tribe Loteae) and  M. truncatula  represent thetwo best-characterized legume genomes. Although there are noimportant crop legumes within the Loteae, the relatively recentdivergence and sister-clade relationship to the galegoid legumes(Fig. 1) offers a potentially useful point of comparison to  M.truncatula . We determined that  M. truncatula  and  L. japonicus share a remarkably high level of conserved macrosynteny,dominated by a few large chromosome arm-size rearrangements.The availability of fully sequenced large insert clones [i.e., BACsand transformation-competent BAC (TACs)] at each of thesegenetically syntenic loci provided an opportunity to evaluate thecorrelation between genetic macrosynteny and sequence mi-crosynteny.Conservedmicrosyntenywascharacterizedby  80%of close homologs in the same order and transcriptional orien-tation, similar to values obtained between human and mouse(30) and within the range identified for the grasses (7). Thecurrentanalysisalsorevealssignificantdivergencebetweenthesetwo legume genomes, with the insertion or deletion of individualor groups of genes accounting for   20% divergence of genecontent in microsyntenic intervals. Species-specific tandem du-plication of genes accounted for an additional 12–17% diver-gence of gene content, and each species possessed a uniquedistribution of mobile DNAs. Of 21 tandemly duplicated genes,only one duplication was reciprocal. Similarly, of 26 cases of mobile DNAs, only one mobile DNA was potentially syntenic.The lack of ancestral tandem duplication is suggestive of eitherefficient removal of tandem duplicates that predate speciation ora recent increase in the rate of tandem duplication. Moreover,the observation that tandemly duplicated genes are occasionallyinterspersed with single copy genes (Table 4) suggests thatpurification of duplicates by homologous recombination wouldsimultaneously eliminate the intervening single copy gene(s).Such a mechanism could explain, at least in part, the loss of genehomologs from microsyntenic regions.Syntenic relationships were significantly more convolutedbetweenthegalegoidandphaseoloidclades.Twenty-fivepercentof genetically mapped orthologous genes were potentially non-syntenic, resulting in smaller regions of colinearity than thoseobserved between  M. truncatula  and  Pi. sativum  or between  M.truncatula  and  L. japonicus . This fragmentation of synteny mightbe expected based solely on the differences in chromosomenumber between these two clades. Despite the relatively largenumber of genetic markers used for comparison, synteny be-tween  M. truncatula  and soybean was difficult to characterize.Weattributethissituationtoacombinationofrecentduplicationand low rates of polymorphism in the soybean genome. Never-theless, comparison of putatively orthologous BAC clone pairs Fig. 5.  Consensus comparative map data for six legume species. Speciesabbreviations are as in Fig. 2. S and L denote short and long arms of eachchromosomein M.truncatula (20).Syntenyblocksaredrawntoscalebasedongenetic distance. Solid lines, postulated rearrangements; double-headed ar-rows, postulated inversions. Gene-specific markers that disrupt synteny areS- SHMT  , R- RNAH  , T- TRPT  , M- MMK1 , P- PTSB , A-  ARG10 , D- 6DCS  , and E- REP  . Choi  et al  . PNAS    October 26, 2004    vol. 101    no. 43    15293       A      G      R      I      C      U      L      T      U      R      A      L      S      C      I      E      N      C      E      S
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