a sequence-based genetic map of medicago truncatula and

Copyright 2004 by the Genetics Society of America

A Sequence-Based Genetic Map of Medicago truncatula and Comparison ofMarker Colinearity with M. sativa

Hong-Kyu Choi,*,† Dongjin Kim,* Taesik Uhm,† Eric Limpens,‡ Hyunju Lim,* Jeong-Hwan Mun,*Peter Kalo,§,** R. Varma Penmetsa,* Andrea Seres,§ Olga Kulikova,‡ Bruce A. Roe,††

Ton Bisseling,‡ Gyorgy B. Kiss§,** and Douglas R. Cook*,1

*Department of Plant Pathology, University of California, Davis, California 95616, †Molecular and Environmental Plant Sciences Program,Texas A&M University, College Station, Texas 77843, ‡Department of Plant Sciences, Wageningen University, 6703HA Wageningen,

The Netherlands, §Biological Research Center, Institute of Genetics, H-6701 Szeged, Hungary, **Agricultural Biotechnology Center,Institute of Genetics, H-2100 Godollo, Hungary and ††Advanced Center for Genome Technology,

University of Oklahoma, Norman, Oklahoma 73019

Manuscript received August 18, 2003Accepted for publication December 12, 2003

ABSTRACTA core genetic map of the legume Medicago truncatula has been established by analyzing the segregation

of 288 sequence-characterized genetic markers in an F2 population composed of 93 individuals. Thesemolecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs,covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primersdesigned to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms weresignificantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to maptranscribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, whichhave been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. Weanticipated that mapping of conserved coding regions would have utility for comparative mapping amonglegumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences acrossa range of legume species. As an initial test of this strategy, we used primers designed against M. truncatulaexon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basisfor a Medicago composite map.

THE genus Medicago contains in excess of 54 charac- Mediterranean basin and agronomic use particularly interized species (Lesins and Lesins 1979; Small Australia, M. truncatula has great potential for the study

and Jomphe 1988), with the majority of species being of both basic and applied aspects of plant biology. Theeither diploid annuals or tetraploid perennials. The natural attributes of M. truncatula that make it desirablemost important economic species of Medicago is the as an experimental system include its annual habit, dip-tetraploid perennial Medicago sativa, or alfalfa, although loid and self-fertile nature, abundant natural variation,several annual Medicago species are of regional agricul- relatively small 500-Mbp genome, and close phyloge-tural importance either as forage crops or for intercrop- netic relationship to the majority of crop legume speciesping as a means to enhance soil nitrogen. M. truncatula (Barker et al. 1990; Cook 1999). Moreover, over theis native to the Mediterranean basin, where the exis- past decade several research groups have developed thetence of numerous native populations has provided an tools and infrastructure for basic research, includingimportant resource for population biology and surveys efficient transformation systems (Trieu and Harrisonof natural phenotypic variation (Bonnin et al. 1996a,b). 1996; Trinh et al. 1998; Kamate et al. 2000), collectionsIn addition to its native distribution, M. truncatula has of induced variation (Penmetsa and Cook 2000), well-been cultivated for close to 1 century in Australia, where characterized cytogenetics (Cerbah et al. 1999; Kuli-it was developed on a limited scale as a winter forage kova et al. 2001), and a collaborative research networkand for use in ley rotation with wheat (Davidson and (http://www.medicago.org). Research efforts on M.Davidson 1993). Also known by the common name truncatula encompass a broad range of issues in plant“barrel medic,” M. truncatula is well suited as a crop in biology, ranging from studies of population biologyareas of nonacidic soils and low winter rainfall. (Bonnin et al. 1996a,b) and resistance gene evolution

As a consequence of its native distribution in the (Cannon et al. 2002; Zhu et al. 2002) to the molecularbasis of symbiotic interactions (e.g., Penmetsa and Cook1997; Catoira et al. 2000, 2001; Harrison et al. 2002;

1Corresponding author: Department of Plant Pathology, University of Ben Amor et al. 2003; Limpens et al. 2003; Liu et al. 2003;California, 1 Shields Ave., Davis, CA 95616.E-mail: [email protected] Mathesius et al. 2003) and micronutrient homeostasis

Genetics 166: 1463–1502 ( March 2004)

1464 H.-K. Choi et al.

(Nakata and McConn 2000; McConn and Nakata variable 5S rDNA loci and two ESTs that appear tohave been the focus of lineage-specific expansion or2002; Ellis et al. 2003). Of importance to these hypothe-

sis-driven investigations is the parallel development of contraction.tools for genome analysis, including a large collectionof expressed sequence tags (ESTs) and an ongoing phys-

MATERIALS AND METHODSical map and whole-genome sequencing effort, as wellas corresponding activities on metabolic profiling and Identification of expressed sequence tags for genetic

marker development: M. truncatula EST sequences were ob-proteomics.tained from the National Center for Biotechnology Informa-A key resource for both classical genetic and genomicstion (NCBI) dbEST and used to query the NCBI databases

efforts in M. truncatula is a genetic map composed of using blastx, blastn, or tblastx. M. truncatula ESTs with highwell-characterized molecular markers. Thoquet et al. similarity to genes discovered in other organisms (principally

Arabidopsis and/or other legumes) were selected for further(2002) have produced a genetic map based primarilyanalysis. Analyses were conducted against public domain se-on the analysis of anonymous sequence polymorphismsquences available at NCBI in February 2000. In the initial[i.e., amplified fragment length polymorphism (AFLP)attempt, we screened �2700 M. truncatula ESTs using blast

and randomly amplified polymorphic DNA (RAPD) and selected 274 ESTs as marker candidates. Oligonucleotidemarkers]. However, the increasing sequence informa- primers were designed from predicted exon sequences using

the Lasergene PrimerSelect software package (DNAStar, Madi-tion for M. truncatula provides an opportunity to mapson, WI) with the following general guidelines. In cases insequence-characterized loci. We have used such a strat-which introns could be predicted by aligning an M. truncatulaegy in previous studies to describe the organization andEST with a corresponding genomic sequence of Arabidopsis,

distribution of resistance gene analog sequences (Zhu primer pairs were designed to anneal in exon sequences andet al. 2002) and as the basis for examining genome to amplify across intron regions. In cases in which an M.

truncatula EST possessed similarity to sequences identified inconservation between M. truncatula and Arabidopsis thali-other legumes (on the basis of blastn), sequence alignmentsana (Zhu et al. 2003).were used to design oligonucleotide primers that would am-The goal of the current study was to develop codomi-plify DNA fragments from each of the corresponding legume

nant genetic markers for the transcribed region of the genomes. The soybean database contributed most of the le-M. truncatula genome. Ancillary goals included provid- gume sequences for sequence comparison due to the relative

abundance of soybean ESTs, and thus a majority of the ESTing a community resource for genetic mapping in M.primer pairs amplify sequences from the soybean genometruncatula and developing a set of conserved genetic(H.-K. Choi and D. Cook, unpublished results).elements for comparative map analysis within the Faba-

Identification of BAC clones for genetic marker develop-ceae. We have emphasized the development of se- ment: RFLP probes previously mapped in crop legumes werequence-based genetic markers, as these are anticipated used to identify homologous M. truncatula BAC clones on the

basis of DNA hybridization. Soybean RFLP clones with highto have wider application among populations within ahomology to genes in the NCBI database (May 1999) basedspecies and between related species. Toward this end,on blastx were selected as probes for Southern blot analysis.we used the extensive collection of ESTs for M. trunca-High-density filters containing five times the coverage of the

tula (e.g., Fedorova et al. 2002) to develop genetic mark- M. truncatula genome were obtained from the Clemson Uni-ers for genes that exhibit high sequence conservation versity Genome Center and hybridized with [32P]dCTP-labeled

probes essentially as described by Nam et al. (1999). Putativewith other legumes or with Arabidopsis. In parallel topositive clones were retrieved from the BAC library, purified,the EST approach, we used DNA hybridization and se-and used for DNA isolation by means of the QIAGEN (La Jolla,quence information to identify and genetically map bac-CA) plasmid kit according to the manufacturer’s instructions.

terial artificial chromosome (BAC) clones containing Purified BAC DNA was digested with HindIII, resolved in agenes of special interest [e.g., M. truncatula resistance 0.6% agarose gel, and used for a second round of Southern

blot analysis. Hybridization patterns were used to confirm thegene analogs, genes expressed during symbiosis, or ho-original hybridization result and to distinguish paralogousmologs of mapped soybean restriction fragment lengthloci on the basis of the size of the hybridizing band and thepolymorphism (RFLP) clones]. The majority of the ge-correspondence between BAC fingerprints. The resulting BAC

netic markers (BAC and EST) are anchored to BAC clones were end sequenced using oligonucleotide primers thatclone contigs, providing an important opportunity to are complementary to the BAC clone polylinker: SQ-BAC-L

(5�-AACGCCAGGGTTTTCCCAGTCACGACG-3�) and SQ-use fluorescence in situ hybridization (FISH) to resolveBAC-R (5�-ACACAGGAAACAGCTATGACCATGATTACG-3�).ambiguities in the genetic map, as well as to increaseTwenty-microliter sequencing reactions contained 500 ng ofthe integration of genetic, cytogenetic, and physicalBAC DNA, 8 �l of ABI BigDye (Perkin-Elmer, Norwalk, CT),

map data. The resulting genetic map defines eight link- and 5 pmol of primer. Sequencing reactions were performedage groups (LGs), corresponding to the eight cytogenet- with a 2-min initial denaturation step at 97�, followed by 40

cycles at 97� for 6 sec and 60� for 5 min. On the basis of BACically defined M. truncatula chromosomes (Kulikova etend sequence information, oligonucleotide primer pairs wereal. 2001). To test the utility of these genetic markersdesigned to PCR amplify the corresponding genomic DNAfor cross-species comparison, we analyzed 68 sequence-fragment from M. truncatula mapping parents, genotypes A17

based markers in a diploid M. sativa (alfalfa) population. and A20.The results demonstrate that the two species are essen- Identification of polymorphic sequences and marker devel-

opment: Parental genomic DNAs (Mt A17 and Mt A20) weretially colinear, with the exception of the notoriously

1465M. truncatula Genetic Map

amplified by the polymerase chain reaction using oligonucleo- and to develop CAPS markers (as described above for M.truncatula). In cases in which CAPS markers could not betide primers designed from ESTs or BAC end sequences, as

described above. Ten-microliter PCR reactions contained the developed, alleles were scored in F2 populations by directsequencing of the PCR products. In such cases, a limitedfollowing reagents: 20 ng of genomic DNA template, 1� PCR

reaction buffer, 2.5 mm MgCl2, 0.25 mm of each dNTP, 5 pmol number of F2 individuals were selected to provide fine discrimi-nation within the desired genetic interval, aided by a color-of each primer, and 0.5 unit of HotStarTaq DNA polymerase

(QIAGEN). PCR thermocycling reactions were performed coded genotype map of the diploid alfalfa population (Kisset al. 1998). In a typical mapping experiment, 138 M. sativawith a 15-min initial denaturation/activation step, followed

by 35 cycles at 94� for 20 sec, 55� for 20 sec, and 72� for 2 F2 individuals were analyzed. The F2 mapping population wasderived from a single F1 plant (F1/1), based on a cross betweenmin, with a final extension step of 5 min at 72�. PCR products

were assessed by gel electrophoresis in 1% agarose, visualized the diploid yellow-flowered M. sativa ssp. quasifalcata and thediploid purple-flowered M. sativa ssp. coerulea (described byby means of ethidium bromide staining. PCR reactions pro-

ducing single bands were selected for sequencing using an Kiss et al. 1993).Genetic distances were calculated by the “classical” max-ABI377 or ABI3730XL automated sequencer and the ABI

PRISM BigDye terminator sequencing ready reaction kit (Per- imum-likelihood method using MAPMAKER/EXP 3.0(Lander et al. 1987; Lincoln et al. 1992). Linkage was deter-kin-Elmer). Sequencing reactions of 10-�l volume contained

10–50 ng of PCR amplicon, 4 �l of ABI BigDye reagent, and mined by the “Group” command set at LOD 3.5 and a distanceof 40 cM based on the Kosambi mapping function. The order5 pmol of primer. Sequencing thermocycling was performed

with a 1-min initial denaturation step at 96�, followed by 35 of the markers was determined by the “Order” command(LOD 3.0, � � 0.40). Raw genotype data were checked usingcycles at 96� for 10 sec, 55� for 5 sec, and 60� for 4 min. DNA

sequence alignments, produced with the Sequencher 3.1.1 the color mapping method as described by Kiss et al. (1998).Color mapping provides a convenient means to visually inspectprogram (Gene Codes, Ann Arbor, MI), were used to survey

the parental alleles for polymorphic sites. Length and codomi- and curate genotypes for each individual of the population,thereby identifying potential genotyping errors and rare re-nant polymorphisms could be assayed directly by means of

agarose gel electrophoresis. Single-nucleotide polymorphisms combination events, and to propose linkage or nonlinkage.Identification of BAC clones for FISH analysis: In cases in(SNPs) were converted to cleaved amplified polymorphic se-

quences (CAPS) by identifying SNPs that confer differential which BAC clones were not previously identified by means ofDNA hybridization, we used the polymerase chain reaction torestriction enzyme sites between the two parental alleles

(Konieczny and Ausubel 1993; Hauser et al. 1998; Michaels identify candidate BAC clones. BAC DNA pools were con-structed either from the 5� coverage BAC library, as describedand Amasino 1998). In cases in which a suitable restriction

enzyme site was not identified, oligonucleotide primers with by Nam et al. (1999), or from a more recently developed 20�coverage BAC library of M. truncatula (D. Kim and D. R. Cook,a single nucleotide mismatch were designed adjacent to the

polymorphic position, such that a restriction site was created personal communication). Candidate BAC clones were puri-fied and cultured overnight on Luria broth agar mediumin the PCR product of one parent, but not the other (so-

called derived CAPS markers, or dCAPS; e.g., Neff et al. 1998). supplemented with 30 �g/ml of chloramphenicol. The iden-tity of BAC clones was confirmed by PCR, with amplified prod-Genotyping and data analysis: Plant genomic DNA was iso-

lated using the DNeasy plant mini kit (QIAGEN) according ucts assessed for size and intensity by means of gel electropho-resis in 1% agarose.to protocols provided by the manufacturer. Two parental lines

of M. truncatula, Jemalong A17 (the primary experimental FISH with BAC clones on prometaphase and pachytenechromosomes: Anthers of M. truncatula A17 flower buds weregenotype used in most investigations to date) and A20, were

chosen previously (Penmetsa and Cook 2000) to facilitate used for producing mitotic prometa-phase (tapetum) and mei-otic pachytene chromosome spreads. A detailed descriptiongenetic mapping and subsequent map-based cloning of genes

defined by their mutant phenotype. The basic mapping popu- of the chromosome preparation procedure and FISH is pro-vided by Kulikova et al. (2001). BAC DNA used as probeslation consisted of 93 F2 progeny derived from a cross of A17

and A20. In regions of specific interests, or where additional was isolated according to the alkaline lysis method and labeledwith either biotin-16-dUTP or digoxigenin-11-dUTP using arecombinants were desired to establish marker order, up to

120 individuals were genotyped. nick-translation mix (Roche). In some cases, BACs were la-beled with a mixture of both dUTPs (in ratio 1:1) to produceFor purposes of marker genotype analysis, the F2 DNAs were

analyzed in parallel with three control DNAs (A17 maternal yellow FISH signals after detection. Two to five probes wereused simultaneously in each hybridization, including BACshomozygous line, A20 paternal homozygous line, and hetero-

zygous DNA) in a structured 96-well microtiter plate format. that were mapped previously (Kulikova et al. 2001) and servedas landmarks for individual chromosomes.Briefly, following PCR �50–100 ng of product (1–2 �l) was

transferred to a new 96-well plate containing 1–5 units of a Biotin-labeled probes were detected with avidin-Texas redand amplified with biotin-conjugated goat-antiavidin andpredetermined restriction enzyme (Table 1) in a total volume

of 8 �l. Digestion was carried out at the manufacturer-specified avidin-Texas red (Vector Laboratories, Burlingame, CA). Di-goxigenin-labeled probes were detected with sheep-antidigoxi-temperature for 2–4 hr. Cleaved DNA fragments were analyzed

by agarose gel electrophoresis and genotypes were recorded genin fluorescein-5-isothiocyanate (FITC; Roche) and ampli-fied with rabbit-anti-sheep FITC ( Jackson ImmunoResearchas follows: homozygous maternal (A17) as “A,” homozygous

paternal (A20) as “B,” heterozygous as “H,” not A as “C,” not Laboratories, West Grove, PA). Chromosomes were counter-stained with 4�,6-diamidino-2-phenylindole (DAPI) in Vecta-B as “D,” and missing data as “—.”

For M. sativa, genetic marker candidates were first scored shield antifade solution (Vector Laboratories) of 5 �g/ml.Some chromosome preparations were reused for FISH withfor polymorphisms in the parental plants (Mscw2 and Msq93)

and their F1 progeny (F1/1). Markers that displayed easily a new set of probes according to the method of Heslop-Harrison et al. (1992). Images were captured for each fluo-scored polymorphisms (e.g., length variation, dominant inheri-

tance, or heteroduplex formation) were genotyped directly rescent dye separately with a cooled CCD camera system (Pho-tometrics, Tucson, AZ) on a Zeiss Axioplan 2 fluorescenceby means of agarose gel electrophoresis. In cases in which

alleles could not be scored directly on agarose gels, the ampli- microscope, pseudocolored, and merged by means of a Cyto-Vision workstation (Applied Imaging). To separate individualfication products were sequenced to identify polymorphisms


chromosomes, each chromosome was digitally excised and a single locus in M. truncatula. On the basis of similarcopied into a new image using Adobe Photoshop 6.0 (Adobe). reasoning, the remaining soybean RFLP clones identi-

fied either two (13%) or three loci (14%). These resultsare likely to represent an underestimate of gene copy

RESULTSnumber in M. truncatula, as not all BAC clones identifiedby a given probe were subjected to Southern blot analy-Development of genetic markers: With the goal of

constructing a core genetic map of M. truncatula en- sis. The corresponding BAC clones were end sequencedand the information was used to develop 60 geneticriched with gene-based genetic markers, we focused on

three distinct classes of sequences: (1) ESTs with high markers. On a more limited scale, RFLP clones pre-viously mapped in alfalfa or pea were also used to screenhomology to genes known in Arabidopsis and/or other

legume species, (2) M. truncatula BAC clones with high the M. truncatula BAC library for homologous loci andto develop genetic markers on the basis of a similarhomology to mapped soybean RFLP probes, and (3)

genes of predicted function. Table 1 provides a com- strategy. In cases in which RFLP clones from other spe-cies were used to identify M. truncatula BAC clones andplete list of all marker information used in this study.

ESTs with similarity to Arabidopsis and legumes: To iden- derived genetic markers, their species affiliation is listedin Table 1.tify M. truncatula ESTs with high similarity to genes in

other legumes or Arabidopsis, we used BLAST (Alt- Markers developed from sequences of predicted function: Asa counterpart to selecting genes on the basis of BLASTschul et al. 1990) to search the NCBI nonredundant

(nr) and EST (dbEST) databases for related sequences, analysis or DNA hybridization, genetic markers were alsodeveloped from sequences selected on the basis of theirusing the following minimum criteria: tblastx against

nr, �e -50; blastn against dbEST to identify ESTs from presumed function. The largest class of this marker typerepresents the nucleotide binding site-leucine-rich repeatother legume species (principally the soybean EST data

set), �e -45; and blastn against the Arabidopsis genome superfamily of resistance gene analogs (see Zhu et al. 2002for a comprehensive analysis). An additional 12 genessequence, �e -30. Where possible, sequences were cho-

sen to represent apparently low- or single-copy-number were selected for mapping on the basis of their possiblerole in plant-microbe interactions, including symbiotic ni-genes, using the Arabidopsis genome as a reference

for gene copy number. In total, 141 EST-based genetic trogen fixation (e.g., leghemoglobin, ENOD40, ENOD16,and rip1) and pathogenic associations (e.g., homologsmarkers were developed on the basis of this approach.

BAC clones with homology to mapped RFLP probes mapped of plant chitinase proteins).Identification of polymorphisms and genotyping:from soybean, alfalfa, or pea: In addition to providing a

genetic context for the analysis of M. truncatula genes, Polymorphic loci were identified following PCR ampli-fication and sequencing of alleles from M. truncatulawe desired to produce a framework for comparison of

the genetic maps of related crop legume species. To genotypes A17 and A20, which served as parents of themapping population used in this study (as selected bytest the feasibility of this strategy for soybean (Gylcine

max), a set of 256 publicly available soybean RFLP clones Penmetsa and Cook 2000). Seventeen length and 14dominant polymorphisms were characterized and couldwas purchased from BioGenetic Services and each clone

was sequenced from both ends. The resulting soybean be mapped by virtue of their inherent fragment sizedifferences, or presence/absence criteria, between pa-sequence information was deposited at NCBI as acces-

sion nos. AQ841751–AQ842207 and AQ842113–AQ84- rental alleles. The remaining 257 polymorphisms weresingle-nucleotide differences between parental alleles.2119. A total of 121 of the soybean RFLP clones, �47%

of the sequenced clones, contained a putative open For the majority of SNPs, alleles were converted to CAPSmarkers. SNPs that could not be converted to CAPSreading frame based on BLASTX and TBLASTX

searches of the NCBI database (as of May 1999). These markers were scored by direct sequencing of PCR prod-ucts amplified from DNA of the segregating progeny.putative protein-coding clones were used to screen a

five-times version of the M. truncatula BAC library (Nam In 60 EST markers, PCR primers were designed toanneal in conserved exon regions and to amplify acrosset al. 1999) on the basis of DNA hybridization. DNA was

isolated from the candidate M. truncatula BAC clones, the more highly diverged intron regions. The closestArabidopsis homolog was used to infer intron positiondigested with restriction enzymes, and analyzed follow-

ing agarose gel electrophoresis by Southern hybridiza- and thereby aid primer design. This “intron-targeted”marker strategy assumes that polymorphisms will betion against the corresponding soybean RFLP clones.

This analysis allowed us to verify the original hybridiza- more frequent in intron vs. exon regions. To test thisassumption for the M. truncatula genotypes under analy-tion result and to identify putatively paralogous loci on

the basis of a hybridization fingerprint. In total, 79 of sis, we compiled intron and exon sequences for 47 ofthe intron-targeted markers. Pairwise alignments be-the 121 soybean RFLP probes analyzed in this manner

hybridized strongly to the M. truncatula BAC library. tween the marker genomic sequences and the M. trunca-tula EST data at NCBI allowed us to distinguish exonSeventy-three percent of these 79 soybean RFLP probes

identified only one BAC contig, which we interpret as from intron sequences and to calculate the relative di-


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1227

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Len

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8410

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230

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DK

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8410

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8417

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DK

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AQ

8417

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8420

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9172

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Q84

1815

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Q84

1993

DK

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9170

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NA

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CA

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360

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1948

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8421

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tinue

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8419

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AA

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8421

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AA

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AQ

9170

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CT

TT

AC

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AQ

8419

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8422

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390,

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AA

Q84

1966

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8421

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165

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TT

AQ

8419

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TA

TC

AA

GT

GA

GA

Q84

2154

DK

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AQ

9171

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NA

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AA

AC

CA

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AC

CA

Q84

1966

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2113

DK

293R

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9171

33B

EST

AC

1212

46.1

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IC

APS

80�

210

290

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1917

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AQ

8421

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7136

BE

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380

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GA

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TABLE 2 the estimated correlation between the physical and ge-netic distance is 970 kbp/cM, in practice this value variesIntron analysis for EST markersaccording to the specific regions under analysis, withprevious analyses of five distinct euchromatic chromo-Total no. of loci analyzed 47somal regions yielding values ranging from 200 to 1100Total length analyzed (bp)

Exon 10,693 kb/cM (Ane et al. 2002; Gualtieri et al. 2002; SchnabelIntron 12,599 et al. 2003).

Intron size (bp) A total of 177 codominant markers with completeMinimum/maximum 78 � 747genotype information were designated as “framework”Mean size 161markers (Figure 1). The majority of framework markersTotal no. of polymorphisms

Exon 21 segregated as expected for codominant (1:2:1) alleles;Intron 89 however, 32% (56/177) of the markers exhibited dis-

Average no. of nucleotides/polymorphisms torted segregation, with the expected frequency of het-Exon 509erozygous individuals but overrepresentation of one ho-Intron 142mozygous state and underrepresentation of the other.Polymorphism ratio of exon/intron 1:3.6

No. of mutations in exons In all cases, distorted marker segregation identified re-Synonomous 17 gions of multiple markers with abnormal ratios of al-Nonsynonomous 4 leles. In addition to linkage groups 4 and 8, which are

discussed in greater detail below, three markers (i.e.,ppPF, NCAS, and TUP) on the short arm of chromo-some 1 exhibited an excess of A17 homozygotes; 11vergence of each (Table 2). On the basis of this limited

survey, the average intron size in M. truncatula was 161 contiguous markers on the long arm of chromosome 3(i.e., GSb through DK273L) exhibited an excess of A20bp, with a range of 78–747 bp, and the GT-AG rule

for intron junctions was strictly conserved. As expected, homozygotes; and two markers on the long arm of chro-mosome 7 (i.e., VBP1 and ENOL) exhibited an excesspolymorphisms were more frequent in intron sequences

(on average, 1 SNP every 142 bp) than in the adjacent of A17 homozygotes.In the initial analysis, six well-defined linkage groupscoding regions (on average, 1 SNP every 509 bp), with

80% of exon SNPs predicted to represent synonymous could be identified. These linkage groups were charac-terized by normal Mendelian segregation of marker locichanges. In the case of 40 marker genes, we analyzed

the correspondence between 64 empirically determined (with the exception of the regions noted above), asshown by example for linkage group 2 (Figure 2a). TheM. truncatula introns and the number and position of

introns in the Arabidopsis homologs. We identified only integrity of each of these six linkage groups (i.e., linkagegroups 1, 2, 3, 5, 6, and 7) was confirmed previouslya single discrepancy, namely a first intron in marker

gene ASN2, present in Medicago but absent from the (Kulikova et al. 2001) by FISH studies in which multipleBAC clone probes from each linage group could beArabidopsis homolog (At3g47340). The same first in-

tron was present in six additional legume species (i.e., assigned to a single pachytene chromosome.In contrast to the situation for the six linkage groupsM. sativa, Pisum sativum, Phaseolus vulgaris, Vigna radiata,

Lotus japonicus, and G. max) from which the ASN2 PCR mentioned above, the 55 additional marker loci re-solved unexpectedly into four linkage blocks. A majorityproduct was sequenced (data not shown), indicating

that the intron is ancestral to this group of Papilionoid of these loci exhibited distorted segregation ratios, withan excess of A20 homozygotes and an underrepresenta-legumes.

Genetic map construction: The genetic map shown tion of A17 homozygotes, as shown in Figure 2, b andc. Two lines of genetic evidence suggest that these 55in Figure 1 was derived from the analysis of 274 codomi-

nant and 14 dominant PCR-based genetic markers. In genetic markers belong to two linkage groups. First, wemapped 26 of these loci on the genetic linkage map oftotal, 93 F2 individuals from a cross between M. trunca-

tula ecotypes A17 and A20 were genotyped. A skeleton the closely related M. sativa, where they resolved intotwo well-defined linkage groups (Ms LG4 and Ms LG8,version of this map was used previously to develop an

integrated cytogenetic and genetic map of M. truncatula respectively), as described below. Second, selectedmarker loci from within the distorted regions were geno-genotype A17 (Kulikova et al. 2001), and thus the eight

genetic linkage groups correspond to the individual typed in the M. truncatula segregating population de-rived from genotypes A17 and DZA315 used by Tho-chromosomes, with chromosome numbering derived

from the corresponding linkage groups in M. sativa quet et al. (2002) for construction of an AFLP- andRAPD-rich genetic map. In each case, the markers(Kiss et al. 1993; Kalo et al. 2000), as determined below.

By convention, the cytogenetically determined short mapped to M. truncatula linkage groups that had beenpreviously determined to correspond to the counter-chromosome arms define the top of each linkage group.

The 288 genetic markers span 513 cM with an average parts of M. sativa linkage groups 4 and 8 (G. Kiss,personal communication).distance between markers of 1.8 cM (Table 3). Although


Figure 1.—Core geneticmap of M. truncatula. A totalof 288 molecular markersand 5 phenotypic markerswere positioned on eightlinkage groups, as follows.A total of 177 codominantmarkers are classified as“framework” markers, des-ignated by horizontal linesconnecting to the linkagegroup diagram. Frameworkmarkers shown in bracketscould not be separated ge-netically from the adjacent,leftward framework marker.Markers that were geneti-cally inseparable from morethan one framework marker(e.g., those with incompletegenotype information ordominant markers) areplaced to the right of a verti-cal bar; the position of thevertical bar designates theextent of the genetic inter-val containing the marker.EST-based markers areshown in boldface type;nodulation-related markersare shown in red; BAC end-sequence-tagged (BEST)markers are preceded bythe initials “DK” [with theexception of BEST resis-tance gene analog (RGA)markers]. RGA markers areshown in blue, with the pre-fix “R-” signifying BESTmarkers and “R-EST” signi-fying EST-based markers.

To test the assumption that these markers correspond mosomes. One of these chromosomes, containing34J06, could be identified as chromosome 4 accordingto loci on chromosomes 4 and 8, respectively, of M.

truncatula genotype A17, we used FISH to determine to our knowledge of centromere position and locationof a diagnostic repeat, MtR1, in the pericentromericthe physical location of 16 of these markers in pachytene

chromosome spreads (Figure 3, a–e). As a prelude to heterochromatin of the short arm (data not shown). Afirst series of hybridizations was performed with fivethis analysis, each genetic marker was converted to a

corresponding BAC clone contig by hybridizing PCR BACs, four of which, namely 10F20, 1P05, 5K15, and41H08, were mapped in a previous study (Kulikova etfragments to high-density filters of the M. truncatula

BAC library (Nam et al. 1999) or by PCR analysis of a al. 2001). In a second series of hybridizations, a newset of probes, including 15B23, 06B09, 66M02, 34J06,BAC library DNA multiplex. A total of 16 BAC clones

were used as probes for FISH analysis, as highlighted 47M03, 70L14, and 11C13, was used. All of these BACclones mapped to chromosome 4. The individual hy-in Figure 3, b and c. Initially, we observed that BAC

clones 34J06 and 43B05 gave signals on different chro- bridization patterns are shown in Figure 3, a–c, while a


Figure 1.—Continued.

composite diagram integrating genetic and cytogenetic cies, which allowed the direct application of geneticmarkers in either direction. Of 81 markers analyzed, 68data for linkage group 4 is shown in Figure 3f. On the

basis of a similar set of analyses, five BAC clones, 43B05, were successfully mapped. For the remaining 13 mark-ers, 4 primer pairs failed to amplify M. sativa DNA, 222O13, 69K21, 50M17, and 10M16, were positioned on

chromosome 8. The individual hybridizations are shown markers lacked polymorphism, and 7 markers gener-ated uninterpretable sequence (probably mixtures ofin Figure 3, c–e, with a composite summary of the ge-

netic and cytogenetic data for linkage group 8 shown multiple loci). As shown in Figure 4, the marker align-ment between the two Medicago maps reveals an ex-in Figure 3g.

Comparative linkage analysis between M. truncatula tremely high level of synteny between M. truncatula andM. sativa, including the distorted regions of M. trunca-and M. sativa: Constructing a comparative map between

M. truncatula and M. sativa was facilitated by the high tula linkage groups 4 and 8, described above.Despite the overall high level of similarity, severallevel of nucleotide conservation between these two spe-


TABLE 3

Distribution of marker types by linkage group

No. of markers Distance (cM)

LG Total EST BEST RGA Phenotypic Total Average

1 31 22 7 2 0 64.3 2.012 32 14 15 2 1(dmi1) 60.9 1.973 47 15 14 18 0 78.2 1.784 47 25 11 10 1(sunn) 56.5 1.235 39 16 15 7 1(dmi2) 76.5 1.966 31 8 2 21 0 47.3 1.487 28 15 11 1 1(skl) 54.5 2.028 38 26 5 6 1(dmi3) 75.0 2.08Total 293 141 80 67 5 513.2 1.78

EST, expressed sequence tag marker; BEST, BAC end-sequence-tagged marker; RGA, resistance gene analogmarkers. Phenotypic markers represent nodulation mutations mapped on the basis of the segregation ofnodulation phenotypes in F2 progeny of genotype A17 mutant lines crossed with A20 wild type.

differences were noted. One apparent difference was tion pattern of the NUM1 probe in M. sativa was com-plex, generating 30 bands. The deduced genotypesthe position of a 5S rDNA locus. In M. truncatula, a 5S

rDNA locus mapped to LG5, while in M. sativa a 5S generated at least five polymorphic loci, of which one(Ms-NUM1) mapped to LG4 and the other (Ms-NUM2)rDNA locus was mapped to LG4. However, cytogenetic

analysis indicates the presence of three 5S rDNA loci mapped to LG8. The middle repetitive-like hybridiza-tion patterns of PCT in M. truncatula and of NUM1 inin M. truncatula genotype A17 on LG2, LG5, and LG6

(Kulikova et al. 2001), while the number of 45S rDNA alfalfa suggest that PCT and NUM sequences may haveevolved differently in these two closely related plantloci has been observed to vary between genotypes of M.

truncatula (T. Bisseling and O. Kulikova, personal species.communication). The position and number of 5S rDNAloci has also been observed to vary between ecotypes of

DISCUSSIONA. thaliana (Fransz et al. 1998), so it should not besurprising to find such a difference between species of In this study, we positioned 288 sequence-based mark-

ers on the genetic map of M. truncatula, covering 513the same genus.We noted two additional differences that are likely cM. Each linkage group contained an average of 36

markers, with a range of 27–47 (Table 3). Thoquetto be more substantive than those of the rDNA loci,described above. The PCT primers listed in Table 1 et al. (2002) recently published a genetic map of M.

truncatula that spans 1125 cM and is composed of 289,identified a single locus on M. truncatula linkage group4. However, Southern blot analysis of M. truncatula geno- predominantly RAPD and AFLP, genetic markers. The

difference in total genetic distance covered by the twomic DNA using the PCT PCR fragment as probe identi-fied four putative paralogous sequences that hybridized mapping efforts may derive from inherent differences

in mapping parents [A17 and DZA315 in the case ofto the PCT marker. One of these loci was polymorphicand mapped to linkage group 2 (Figure 4), while the Thoquet et al. (2002) and A17 and A20 in the present

study] and also from the marker types used. Thus, inother three fragments were not polymorphic for theenzymes used. In M. sativa, only one hybridizing locus contrast to mapping expressed genes as codominant

markers, which was the focus of the current study, thewas evident, corresponding to a polymorphic, singlelocus at the syntenic position on linkage group 2 (Figure AFLP and RAPD strategy used by Thoquet et al. (2002)

maps anonymous loci that typically exhibit dominant4). In a second case, the NUM1 gene was mapped toLG4 in M. truncatula by means of NUM1-specific primer inheritance. Although it is likely that the two strategies

surveyed different regions of the genome, both effortspairs. Using the same primers in M. sativa, an �2-kbpnonpolymorphic fragment was amplified. The gel-puri- produced eight well-resolved linkage groups that could

be readily aligned with the eight genetically definedfied fragment was used as a probe to map NUM loci inboth M. truncatula and M. sativa by means of RFLP. linkage groups of diploid M. sativa (Kalo et al. 2000;

Thoquet et al. 2002). Efforts to link the two geneticThe hybridization pattern of M. truncatula identifiedtwo loci, Mt-NUM1 on LG4 and Mt-NUM2 on LG8. The maps of M. truncatula based on simple sequence repeat

markers derived from ESTs and sequenced BAC cloneslocation of the Mt-NUM1 locus on LG4 correspondedto the locus mapped by CAPS. By contrast, the hybridiza- are currently underway.


genome by means of FISH (Kulikova et al. 2001; O.Kulikova and T. Bisseling, personal communication).These results indicate a high level of correspondencebetween euchromatin and transcribed genes, reminis-cent of the relationship observed in A. thaliana where96% of the transcribed genes are contained withineuchromatic regions of the genome (Arabidopsis Ge-nome Initiative 2000). Consistent with this hypothesis,the average predicted gene density for the 92 geneticallymapped and sequenced, EST-containing BAC clonesis �1 gene/6 kbp (B. A. Roe and D. Kim, personalcommunication). Thus, the correspondence of se-quenced BAC clones with genetically mapped loci ex-pands the total number of ESTs and predicted geneson the genetic map to �1800. The accession numbersfor these sequenced BAC clones are given in Table 1.

In addition to the mapping of ESTs or BAC clonesselected strictly on the basis of homology criteria, thegenetic positions of five phenotypic markers associatedwith nodulation, dmi1, dmi2, dmi3, sun, and skl, areshown in Figure 1. Map positions were determined byvirtue of the fact that the genetic markers developed inthis study were used to map the respective loci in F2

populations of mutant A17 � wild-type A20. With theexception of the skl locus (Penmetsa and Cook 1997),located on the long arm of chromosome 7, the maplocations of the other loci have been previously reported(Ane et al. 2002; Endre et al. 2002; Schnabel et al. 2003)and the information is included here for purposes ofintegration. Interestingly, recent evidence from physicalmap data and complete sequencing of a 500-kb BACcontig indicates that dmi1 is immediately adjacent to thetelomere (Ane et al. 2004), and thus this locus defines agenetic and physical terminus of this linkage group. Inaddition to genes implicated in nodulation based onphenotypic criteria, we also mapped several genes whoseFigure 2.—Segregation ratios for markers on linkageexpression patterns are correlated with nodule develop-groups (a) 2, (b) 4, and (c) 8. The primary feature of segraga-

tion distortions on linkage groups 4 and 8 is an overrepresenta- ment or function. Several of these genes, includingtion of genotype A20 homozygotes and a corresponding un- ENOD40 (Yang et al. 1993; Crespi et al. 1994), thederrepresentation of genotype A17 homozogotes. Values on Rhizobium-induced peroxidase (rip1; Cook et al. 1995),the horizontal axis correspond to the genetic position of mark-

and the leghemoglobin gene LB1 (Gallusci et al.ers in the respective Figure 1 linkage groups.1991), map to LG5, which also contains dmi2 (Endreet al. 2002) and the syntenic counterpart of the Sym2region of P. sativum (Gualtieri et al. 2002; Limpens et al.2003). Despite the apparent abundance of nodulation-Because the genetic markers used in this study are

primarily expressed sequences or BAC clones that con- associated genes on linkage group 5, several nodulationgenes (nodule expressed transcripts and phenotypicallytain predicted genes, their position in the genome can

be considered to provide a rough definition of the “gene mutant loci) are distributed elsewhere in the genome,including a cluster of ENOD8-like genes on linkagespace” of M. truncatula. On the basis of cytogenetic anal-

ysis (Kulikova et al. 2001), the structure of M. truncatula group 1 (Dickstein et al. 2002), ENOD16 on linkagegroup 8, a cluster of apyrase genes on linkage group 7chromosomes is apparently relatively simple, with con-

densed heterochromatic DNA in centromeric and peri- (Cohn et al. 2001), and sunn, skl, dmi1, and dmi3 onlinkage groups 4, 7, 2, and 8, respectively.centromeric islands, flanked by mostly euchromatic

arms. In the process of constructing the cytogenetic In addition to genes implicated in symbiosis, 100resistance gene analogs have been previously mappedmap for this species, 60 of the EST-containing BAC

clones genetically mapped in this study also have been to 67 separate loci (Zhu et al. 2002). The majority ofmarkers for toll/interleukin receptor (TIR) and non-mapped to euchromatic regions of the M. truncatula


Figure 3.—Correlation ofcytogenetic and genetic mapsfor linkage groups 4 and 8 ofM. truncatula genotype A17. (aand b) FISH mapping of BACclones on chromosome 4. (c)Simultaneous hybridization withchromosome-specific probes(i.e., 11C13, 43B05, and 50-M17) distinguishes two pachy-tene chromosomes. (d and e)Mapping of BAC clones onchromosome 8. (f and g) Ideo-grams of pachytene chromo-some and genetic linkagegroups of M. truncatula. BACclones are positioned on theideogram according to theirrelative positions in relationto centromeres, which aremarked as “Cen” in the individ-ual panels. For b and e, the in-dividual chromosomes weredigitally separated with image-processing software.

TIR NBS-LRR resistance gene analogs are clustered, observed, with A20 homozygotes significantly overrepre-sented in the populations (Figure 2, b and c). On thewith major clusters identified on the short arm of link-

age group 3 and throughout linkage group 6. Interest- basis of a combination of comparative genetic mappingin M. sativa and analysis of an alternate mapping popula-ingly, in the absence of the resistance gene analog mark-

ers, linkage group 6 contains only 10 genetic markers tion of M. truncatula (Thoquet et al. 2002), we wereable to resolve the genetic relationships between theseand fails to coalesce as a distinct linkage group. Thus,

linkage group 6 is threefold underrepresented in the two linkage groups. FISH analysis with geneticallymapped BAC clones was used to verify the predictednumber of non-RGA genes compared to the seven other

linkage groups, while containing 33% of all mapped marker order and linkage group assignments, whilecolor mapping was used to determine that the recombi-RGA loci. Chromosome 6 is also unusual in the respect

that it is the shortest and most heterochromatic of all nation map was consistent with the interpretations fromthese analyses. The value of 32% distorted marker segre-M. truncatula chromosomes (Kulikova et al. 2001).

The genetic map of M. truncatula was difficult to inter- gation observed in this study is similar to the 25% dis-torted segregation reported by Thoquet et al. (2002).pret for linkage groups 4 and 8. In each of these cases

significant deviation from Mendelian segregation was Moreover, in both studies a cluster of markers with dis-


Figure 4.—Comparativegenetic map of M. truncatulaand M. sativa. The relativeseparation of genetic mark-ers on each linkage groupis correlated with geneticdistance, although centi-morgan scales have beenomitted for simplicity. Ge-netic markers mapped be-tween the two genomes aredesignated by boldface type.

torted segregation was observed on chromosome 3, in- nome of M. truncatula. We anticipate that an EST-basedgenetic map will also have utility for the many map-basedcluding the common marker locus “GSb,” suggesting a

possible contribution from the common parental back- cloning projects currently underway in M. truncatula.Finally, a sequence-based genetic map of M. truncatulaground of genotype A17. By contrast, the distorted

marker segregation for linkage groups 4 and 8 observed should have utility for comparison of genome structurebetween legume species and thus for the characteriza-in this study was not evident in the Thoquet et al. (2002)

analysis, suggesting a possible incompatibility between tion of traits with potential application to agriculturein legumes. We have documented a high degree ofA17 and A20 alleles in these genome regions. In the case

of M. sativa, which is an outcrossing species, segregation conservation in gene content and order between thegenomes of diploid M. sativa (alfalfa) and M. truncatula,distortion is typified by an overabundance of the hetero-

zygous genotype (Kalo et al. 2000). This contrasts with suggesting that the current genetic map and ongoinggenome sequencing of M. truncatula will have significantthe overabundance of paternal (homozygous A20 or

A17) genotypes, described in this study. utility for defining genome organization in cultivatedalfalfa (Brouwer and Osborn 1999). Moreover, we an-The ultimate goal of constructing this genetic map

was to describe structural/genetic features of the ge- ticipate that many of the gene-based genetic markers


Figure 4.—Continued.

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a sequence-based genetic map of medicago truncatula and

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