Chromosomal Map of the Model Legume Lotus japonicus

Corresponding author: Andrea Pedrosa, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria., a9807933{at}unet.univie.ac.at (E-mail)

Communicating editor: V. SUNDARESAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Lotus japonicus is a model plant for the legume family. To facilitate map-based cloning approaches and genome analysis, we performed an extensive characterization of the chromosome complement of the species. A detailed karyotype of L. japonicus Gifu was built and plasmid and BAC clones, corresponding to genetically mapped markers (see the accompanying article by SANDAL et al. 2002 , this issue), were used for FISH to correlate genetic and chromosomal maps. Hybridization of DNA clones from 32 different genomic regions enabled the assignment of linkage groups to chromosomes, the comparison between genetic and physical distances throughout the genome, and the partial characterization of different repetitive sequences, including telomeric and centromeric repeats. Additional analysis of L. filicaulis and its F₁ hybrid with L. japonicus demonstrated the occurrence of inversions between these closely related species, suggesting that these chromosome rearrangements are early events in speciation of this group.

LEGUMES are important worldwide for their use as food and forage. They are also remarkable for their ability to grow independently of externally added reduced nitrogen due to symbiosis with rhizobia. Although studies concerning the molecular basis of the plant role in nitrogen fixation and symbiosis have been reported (SCHERES et al. 1990 ; KOUCHI and HATA 1993 ), fast progress has been achieved only recently with the use of the model legume Lotus japonicus (HANDBERG and STOUGAARD 1992 ; SZCZYGLOWSKI et al. 1997 ; SCHAUSER et al. 1998 ; WEGEL et al. 1998 ).

To facilitate the isolation of new genes by map-based cloning, genetic linkage maps have been established for L. japonicus (HAYASHI et al. 2001 ; SANDAL et al. 2002 ) and a bacterial artificial chromosome (BAC) library has been constructed (N. SANDAL, N. ELLIS and J. STOUGAARD, unpublished results). The map presented here was based on a cross between L. japonicus Gifu and the related species L. filicaulis (SANDAL et al. 2002 ). Until recently, only conventional karyotypes were described for both species (CHENG and GRANT 1973 ). With the increasing interest in Lotus, including a genome sequencing project (CYRANOSKI 2001 ; SATO et al. 2001 ), karyotypes based on the prophase condensation pattern and on fluorescence in situ hybridization (FISH) with rRNA genes were determined for two L. japonicus accessions (ITO et al. 2000 ).

FISH has been used, among other applications, for mapping repetitive sequences and multicopy genes and for correlating genetic linkage groups to chromosomes that could be followed in hybrid lines. FISH has also been useful to compare genetic and physical distances, since it is known that recombination is not uniform along chromosomes and, therefore, these distances correlate poorly (SCHWARZACHER 1994 ; GILL 1995 ). The use of FISH for assigning individual markers to plant chromosomes has been demonstrated (JIANG et al. 1995 ; FUCHS et al. 1998 ; DONG et al. 2000 ; FRANSZ et al. 2000 ; CHENG et al. 2001A , CHENG et al. 2001B ; KULIKOVA et al. 2001 ).

We have used different cytogenetic approaches, including FISH, to develop an extensive chromosomal map of L. japonicus Gifu. Additionally, FISH was used to compare the parental lines L. japonicus Gifu and L. filicaulis and the F₁ hybrid from which the F₂ linkage mapping population was derived. The alignment of genetic and chromosomal maps in several positions along the chromosomes provided an understanding of the map in a genome perspective. Furthermore, chromosomal rearrangements between these two closely related species were demonstrated, which led to the resolution of difficulties with the linkage analysis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material:
L. japonicus Gifu B-129-S9 seeds (STOUGAARD and BEUSELINCK 1996 ) were obtained from the USDA-ARS Regional Plant Introduction Station (Pullman, WA). L. filicaulis B37 seeds were obtained from W. F. Grant's collection. The L. filicaulis x L. japonicus F₁ hybrid plant 4-6 is the parent of the F₂ mapping population and is maintained as cuttings.

Pretreatment and fixation:
Root tips were obtained from seeds germinating on moist filter paper or, in the case of the F₁ hybrid, from plants growing in pots. Root tips were pretreated with 8-hydroxyquinoline (2 mM) for 4–5 hr at 16°, fixed in methanol/acetic acid 3:1 (v/v), and stored in fixative at -20° for up to several weeks.

Feulgen staining and chromosome measurements:
L. japonicus roots were hydrolyzed in 1 N HCl at 60° for 10 min, stained in Schiff's reagent (Merck, Darmstadt, Germany) for 2 hr in the dark at room temperature, and squashed in 45% acetic acid. After removal of the coverslip in a cold plate, slides were made permanent in Euparal.

C-banding:
Root tips were macerated in an enzyme solution [3% (w/v) cellulase "Onuzuka R-10" (Serva Electrophoresis, Heidelberg, Germany) plus 10% (v/v) pectinase (Sigma-Aldrich, Vienna) in 0.01 M citric acid-sodium citrate buffer, pH 4.8] at 37° for 1–2 hr, transferred to a drop of 45% acetic acid, and flamed before squashing. C-banding was performed according to SCHWARZACHER et al. 1980 , except for the final staining, which was done by mounting the slides with 2 µg/ml 4',6-diamidino-2-phenylindole (DAPI) in Vectashield (Vector, Burlingame, CA).

Nuclear DNA measurements:
Nuclear DNA content was estimated by Feulgen densitometry and propidium iodide (PI) flow cytometry. Feulgen densitometry was performed according to GREILHUBER and EBERT 1994 , using Pisum sativum "Kleine Rheinländerin" as standard. Ten nuclei were measured per seedling/slide and three pairs of slides (L. japonicus/P. sativum) were analyzed in each of the four replications. PI flow cytometry was performed according to BARANYI and GREILHUBER 1996 and GREILHUBER and OBERMAYER 1997 , using Glycine max "Polanka" and Zea mays line CE-777 as standards. A total of 5000 nuclei were analyzed in three joint measurements per replication, with two and three replications with G. max and Z. mays, respectively.

Cloning of 5S rRNA gene from L. japonicus:
The 5S rRNA repeats were amplified from genomic DNA of L. japonicus by PCR using the primers 5'-GTGCGATCATACCAGC(AG)(CT)TAATGCACCGG-3' and 5'-GAGGTGCAACACGAGGACTTCCCAGGAGG-3'. The PCR product, 400 bp, corresponding to the dimer form, was purified using the Quantum Prep Freeze 'N Squeeze spin column (Bio-Rad, Vienna), ligated, and transformed using the TA cloning kit (Invitrogen, Groningen, The Netherlands). The insert of one positive clone (D2) was determined by cycle sequencing on an ABI 377 automated sequencer (PE Applied Biosystems, Vienna) and the sequence of one repeat unit was included in the GenBank database (accession no. AY040715).

DNA probes:
The probes used were R2, a 6.5-kb fragment of a 18S-5.8S-25S rDNA repeat unit from Arabidopsis thaliana (WANZENBOCK et al. 1997 ); D2, a 5S rRNA clone from L. japonicus; an Arabidopsis-like telomeric probe, amplified by PCR according to IJDO et al. 1991 using the oligomer primers (5'-TTTAGGG-3')₅ and (5'-CCCTAAA-3')₅; and large plasmid and BAC clones containing inserts with known map position (Table 1). The L. japonicus BAC library (N. SANDAL, N. ELLIS and J. STOUGAARD, unpublished results) was screened using genomic and cDNA clones, corresponding to DNA markers. Southern hybridization to some BAC clones with L. japonicus genomic DNA was performed to identify and exclude the BACs bearing highly repetitive sequences. BACs were confirmed by PCR using specific primers. BAC DNA was isolated using a Plasmid mini kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. All probes were labeled by nick translation (Roche Diagnostics, Vienna; Life Technologies, Vienna) with Cy3-dCTP or Cy3-dUTP (Amersham Pharmacia Biotech, Vienna). R2 and D2 were also labeled with biotin-14-dATP (Life Technologies).

FISH:
Slides were prepared as described for C-banding and selected after staining with 2 µg/ml DAPI in McIlvaine's pH 7.0 buffer. Good preparations were destained in 3:1 (v/v) ethanol-acetic acid (30 min) and absolute ethanol (1 hr) and pretreated as described in PEDROSA et al. 2001 . Chromosome and probe denaturation, post-hybridization washes, and detection were performed according to HESLOP-HARRISON et al. 1991 , except for the stringent wash, which was performed, in subsequent experiments, with 0.1x SSC at 42°. Hybridization mixes consisted of 50% (v/v) formamide, 10% (w/v) dextran sulfate, 2x SSC, and 2–5 ng/µl probe. The slides were denatured for 5 min at 75° and hybridized for up to 2 days at 37°. A total of 10 µg of salmon sperm sheared DNA and 10- to 1000-fold excess L. japonicus C_ot 1 fraction (ZWICK et al. 1997 ) were added to the hybridization mix in few experiments. In those cases, slides were denatured first in 50% formamide and 2x SSC and dehydrated in 96% ethanol for 5 min at -20°. Denatured probe and blocking DNA were reannealed in the hybridization mix for 1–5 hr at 37° before being added to the slides. Biotin-labeled probes were detected using ExtrAvidin-FITC conjugate (Sigma-Aldrich, Vienna) in 1% (w/v) BSA. All preparations were counterstained and mounted with 2 µg/ml DAPI in Vectashield. Reprobing of slides for localization of different DNA sequences on the same cell was performed according to HESLOP-HARRISON et al. 1992 up to three times.

Image analysis:
Photographs were taken on a Zeiss Axioplan (Carl Zeiss, Vienna) equipped with a mono cool view CCD camera (Photometrics, Tucson, AZ). Images from the camera were combined and pseudocolored using spectrum software (IPLab, Fairfax, VA). For construction of the idiogram, 5 well-spread, complete, DAPI-stained metaphases, in which each chromosome arm could be recognized by hybridizing with specific probes, were used. For each chromosome, the total length and the length of its presumable short arm were measured in arbitrary units. Relative lengths and arm ratio were calculated for each chromosome and a mean value was calculated for each pair. The total length of the chromosome complement measured from 10 Feulgen-stained metaphases was used to calculate the length of each chromosome in micrometers. At least 10 metaphases bearing clear signals were photographed in order to calculate the position of a clone. Assignment of a clone to a specific chromosome arm was confirmed by reprobing the slides with a clone of known position. All measurements were performed using the "analyze/measure length" function of the IPLab software. Digital images were imported into Adobe Photoshop version 5.0 for final processing.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Karyotype of L. japonicus:
To establish a chromosomal map of L. japonicus, an idiogram was constructed on the basis of the relative chromosome lengths and arm ratios obtained from DAPI-stained cells in which all chromosome arms could be recognized. Feulgen-stained metaphase plates [ Fig 1(1)] were used to measure the total length of the chromosome complement in micrometers. The complement was divided into three groups: one large (1), three medium (2–4), and two small (5 and 6) metacentric pairs. After C-banding [ Fig 1(2)], heterochromatin was observed predominantly at the pericentromeric region, varying in size among different chromosome pairs. Additional C-positive bands were observed in chromosome 2 in the region corresponding to the nucleolar organizer and at the intercalary position in the long arm of chromosome 4.

View larger version (79K): In this window In a new window Download PPT slide   Figure 1. Metaphase plates of L. japonicus Gifu after Feulgen staining (1) and C-banding-DAPI (2). Chromosomes in 2 are indicated by numbers. Bar, 2.5 µm.

Nuclear DNA content of the species was estimated by Feulgen densitometry and propidium iodide flow cytometry (Table 2). The DNA content ratios obtained between L. japonicus and three standards were used to calculate the ratio between L. japonicus and A. thaliana (DOLEZEL et al. 1998 ). L. japonicus has a genome 3.46-fold larger than that of A. thaliana and, therefore, has a mean 1C value of 432 Mbp, if a 1C value of 125 Mbp (ARABIDOPSIS GENOME INITIATIVE 2000) is assumed for A. thaliana. This estimation is close to the already reported values for L. japonicus (1.0–1.1 pg/2C, GRANT 1995 ; 442.8 Mbp/1C, ITO et al. 2000 ) and was further used for calculating the size of each chromosome in megabase pairs.

In situ hybridization on L. japonicus mitotic chromosomes:
Altogether, DNA clones corresponding to 32 different genomic regions plus a centromeric repeat were used as probes for FISH to L. japonicus chromosomes. Clones were screened out of a BAC library (N. SANDAL, N. ELLIS and J. STOUGAARD, unpublished results) or from libraries using genetic markers distributed over the entire genetic map. Occasionally, subclones were used (Table 1). In addition, the Arabidopsis-like telomeric oligomer repeat was used to generate a telomeric probe by PCR. Eighteen genetically mapped genes plus one genetically mapped BAC end could be used for correlating the linkage and the chromosomal maps.

Some of the BACs, however, showed a strong, dispersed pattern along the chromosomes and the position of those clones could not be determined. Blocking with the C_ot 1 fraction of L. japonicus genomic DNA was not effective for most of the BACs tested. The patterns observed with those BACs were basically of three types: almost uniform along chromosomes, more intense labeling of proximal regions, or more intense labeling of pericentromeric region [ Fig 2(3)]. Most likely, these different patterns reflected the distribution of different repetitive DNA families.

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. In situ hybridization to L. japonicus Gifu chromosomes. (3) BAC 1M10 (Lb) showing pericentromeric distribution. (4) Arabidopsis-like telomeric repeat, with arrows showing some centromeric signals. (5) BAC 41P18 showing centromeric localization (arrows indicate centromeres of chromosome 2). (6–9) Chromosome 1 specific clones: (6) BAC S1-1; (7) GAP2 (BAC 35P5, red) and pZF (green; insertion shows enlargement of a representative chromosome 1; the second chromosome 1 is partly folded in this cell). (8) Lnp (BAC 72G19). (9) Nlp1 (BAC 81J4). (10) 45S rDNA (red) on chromosomes 2, 5, and 6 and 5S (green) rRNA genes on chromosome 2 (minor sites on chromosome 5 are indicated by arrows). Bar, 2.5 µm.

Telomeres and centromeres of L. japonicus chromosomes:
An Arabidopsis-like telomeric probe was generated by PCR and proved to be highly specific and sensitive to detect telomeres of Phaseolus vulgaris, another legume species (A. PEDROSA, unpublished data). However, in the case of L. japonicus, signals were always weak and never detected in all chromosome ends. Furthermore, additional centromeric signals were observed with the same intensity and frequency as the telomeric ones [ Fig 2(4)]. When the BAC library was screened with the telomeric oligonucleotides and three of those clones were hybridized to L. japonicus chromosomes, none showed hybridization to the telomeres. However, all showed labeling of centromeres, which was less intense for chromosome 2 than for the others [ Fig 2(5)].

Sequencing of both ends of two of those BACs revealed the presence of a repeated DNA family, named Ljcen1 (GenBank accession no. AF390569). A 440-bp fragment of Ljcen1 showed the same centromeric distribution. Variation among the four sequenced segments was restricted to 1 or 2 bp out of 600 bp. The repeats do not show significant homology to the telomeric DNA or to any sequence in the EMBL/GenBank database, but the telomeric oligonucleotide hybridized to large BamHI fragments that also contained Ljcen1. Furthermore, one centromeric BAC labeled the telomeres of P. vulgaris (A. PEDROSA, unpublished data).

Mapping of L. japonicus chromosomes:
Clones bearing unique sequences enabled the alignment of genetic markers onto chromosomes. Examples of hybridization of unique sequences to each chromosome are shown in Fig 2(6–10) and 3(11–15). The identification of the chromosome arm bearing the signals was based on the relative chromosome length, centromeric position, and heterochromatin pattern and confirmed by rehybridization with previously assigned clones. The 45S rRNA probe was particularly useful for identification of chromosomes [ Fig 2(10)]. Chromosome 2 bears the major, active rDNA site. Chromosome 6 possesses the second largest site and can be easily distinguished from chromosome 5, which bears a minor site, difficult to detect and not reported before (ITO et al. 2000 ). Although neither 45S nor 5S rRNA gene clusters could be mapped genetically, they could be assigned to linkage groups after both maps were correlated.

Positioning of the clones along chromosomes was based on means of several measurements (Table 1). All clones were assigned to a unique chromosome position, except for clone Nlp2, which, in a subset of cells, was detected on chromosome 5 instead of 3, suggesting the presence of a translocation in some individuals. With the exception of chromosome 5, a minimum of two mapped markers per chromosome were assigned to confirm the orientation of genetic vs. chromosome map.

Marker alignment was particularly useful for chromosome 1, because segregation distortion provided two linkage groups instead of one (IA and IB) and additional unlinked markers. We mapped at least two markers per linkage group, plus two unlinked markers, and demonstrated that all are derived from chromosome 1, with unlinked markers residing in the region that connects linkage groups IA and IB [ Fig 2(6–9)].

Physical distances:
A summary with the karyotype data, the position of all mapped clones, and the correlation to the genetic map (SANDAL et al. 2002 ) is given in Fig 4 and in Table 3. The relative distances between the clones in relation to the chromosome length could be compared with their distances on the genetic map. It is clear that these distances often did not correlate.

View larger version (42K): In this window In a new window Download PPT slide   Figure 3. In situ hybridization to the chromosome complement of L. japonicus Gifu (11–15), of L. filicaulis (17–18), and of the F1 hybrid (19). (11) sym13 (936-5.3, red) together with the major rDNA site (green) on chromosome 2. (12) Nlp2 (BAC 92J16, red) and har1-3 (p3.2, green) on chromosome 3 (one chromosome 2 is missing). (13) Myo2 (BAC 60D1, red) on chromosome 4, while chromosomes 2 and 6 are identified by rDNA sites (green). (14) Subclone 71 from BAC 1M10 (Lb) together with clones Lb 112 and 123 (red) on chromosome 5 and rDNA (green) indicating chromosomes 2 and 6 (one chromosome 4 is missing). (15) Gln1 (no. 431, red) together with the rDNA (green) on chromosome 6. (16) 45S rDNA (red) on chromosomes 2 and 6 and 5S (green) rRNA genes on chromosome 2 of L. filicaulis. Centromeres are easily distinguishable by the presence of brighter heterochromatin. Arrows indicate centromeres of acrocentric chromosome. (17) Nlp2 (BAC 92J16, red) on acrocentric chromosome 3 and BAC 10F21 (green) at centromeres of all chromosomes. Arrows indicate centromeres of chromosome 2. (18) Nlp1 (BAC 81J4, red) and Cyc2 (BAC 53C15, green) on chromosome 1 of L. japonicus (J) and L. filicaulis (F). Bar, 2.5 µm.

View larger version (15K): In this window In a new window Download PPT slide   Figure 4. Idiogram of L. japonicus Gifu in comparison to its genetic linkage map established by SANDAL et al. 2002 . Idiogram shows relative chromosome length, position of centromeres, approximate distribution of C-banding heterochromatin, and mapping of plasmid and BAC clones. Clones are either named directly (left) or indicated by the name of the corresponding gene, when genetically mapped (right). Clones in light shading were tentatively localized. Parentheses represent the alternative position of clone Nlp2 in some cells (see RESULTS). Chromosome 6 was used for normalizing chromosome and linkage group lengths. Dotted line in the genetic map indicates a region of uniparental inheritance (see SANDAL et al. 2002 ). Segments bordered by dotted brackets in chromosomes 1 and 3 indicate putative regions inverted between L. japonicus and L. filicaulis.

Chromosomes 1, 5, and 6 seemed to be well covered in the genetic map, with mean ratios of 0.77, 1.03, and 1.05 Mbp/cM, respectively. Chromosomes 2, 3, and 4, on the other hand, showed ratios of 1.42, 1.81, and 2.07 Mbp/cM, respectively, suggesting either suppression of recombination or lack of certain chromosome segments in the genetic map.

When subchromosomal regions were analyzed in more detail, major differences in recombination frequencies along chromosomes were detected (Table 3). In subtelomeric regions of chromosome 1, comprising around 23% of the chromosome length, the ratio between physical and genetic distances was 0.36 Mbp/cM in the short arm (region comprising from the top of the chromosome to the s1-1 gene) and 0.31 Mbp/cM in the long arm (from Lnp to the bottom). Along the central part of chromosome 1, however, the ratio increases to 1.3 Mbp/cM. The major part of linkage group II comprises 30% of the distal part of the long arm of chromosome 2 (from sym13 to the bottom). In this region, the physical vs. genetic ratio is 0.52 Mbp/cM, in contrast to 6.64 Mbp/cM along the rest of the chromosome. In chromosome 3, a lower ratio was again observed for the terminal region of the long arm (0.83 Mbp/cM, from sym8 to the bottom), in contrast to a ratio of 3.04 Mbp/cM for the remaining 75% of the chromosome. Chromosome 4 showed a particularly strong suppression of recombination on its middle part, comprising the centromeric region and an intercalary heterochromatic block. Altogether, except for the rRNA gene clusters on the short arm of chromosome 2, all linkage groups seem to cover the chromosome ends, and lower levels of recombination were observed mainly on proximal chromosome regions.

To check whether chromosome rearrangements between the two parental species were involved in segregation distortions, repression of recombination, and shortening of the genetic map length, the chromosome complement of L. filicaulis and of the F₁ hybrid that served as a source for the mapping population were also analyzed.

Chromosomal analysis in L. filicaulis and in the F₁ hybrid:
After DAPI staining, the chromosome complement of L. filicaulis proved to be similar to the L. japonicus complement. However, the intercalary heterochromatic band on chromosome 4 was not observed and one of the two largest midsized chromosome pairs was acrocentric, as was confirmed by hybridization with a centromeric BAC. Using this clone, the same centromeric pattern was observed, only slightly more dispersed toward the arms. This correlates to the higher content of pericentromeric heterochromatin in L. filicaulis, as inferred from its C-banding-like pattern observed after FISH [ Fig 3(16)].

Hybridization with the 45S and 5S rRNA probes revealed that L. filicaulis chromosome 2 was metacentric [ Fig 3(16)]. On the other hand, two genes that hybridized to the long arm of L. japonicus metacentric chromosome 3 hybridized, in the same orientation, to the end of the long arm of L. filicaulis acrocentric chromosome 3 (data not shown). Furthermore, Nlp2, which in L. japonicus hybridized to the short arm of chromosome 3 [see Fig 3(12)], hybridized to the middle of the long arm of the same chromosome in L. filicaulis [ Fig 3(17)]. In the F₁ hybrid cells, both locations of Nlp2 were observed. This indicates that a large pericentric inversion involving 50% of chromosome 3 distinguishes L. filicaulis from L. japonicus.

Although no morphological alteration was observed in chromosome 1, the high segregation distortion that led to the separation of linkage group I prompted us to analyze the localization of Nlp1 and Cyc2, which were mapped between IA and IB, in the F₁ hybrid [ Fig 3(18)]. Due to the higher amount of pericentromeric heterochromatin in L. filicaulis, both chromosomes 1 could be distinguished.

The distance between the two clones was highly significant in the L. japonicus chromosome, but not significant in L. filicaulis (Table 4). Nlp1 seems to have maintained the same relative position in the two species, whereas Cyc2 was significantly more distal on the L. japonicus than on the L. filicaulis chromosome. Since the difference in total length of chromosome 1 was not highly significant, we favor the explanation that a paracentric inversion, not an insertion/deletion, is responsible for this deviation. Moreover, preliminary meiotic analysis of the F₁ hybrid showed the occasional formation of an anaphase I bridge between two large chromosomes (data not shown), as is typical for paracentric inversions.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have constructed a detailed chromosome map of L. japonicus Gifu, integrating the position of BAC and plasmid clones from 32 genomic regions. Nineteen of these clones were also mapped genetically. This makes the L. japonicus map reported here one of the most extensive correlations of genetic and chromosomal maps in plants, enabling the determination of physical and genetic distance ratios along the whole chromosome complement. Furthermore, comparison to the closely related species L. filicaulis revealed at least two inversions between the genomes, giving insights into the karyotype evolution of the group.

FISH to mitotic and pachytene chromosomes has been previously demonstrated to be very useful in plants for assigning linkage groups to chromosomes (FUCHS et al. 1998 ; DONG et al. 2000 ; CHENG et al. 2001A ; KULIKOVA et al. 2001 ) and for establishing cytogenetic-based physical maps for single chromosomes and chromosome regions (ZWICK et al. 1998 ; ZHONG et al. 1999 ; CHENG et al. 2001B ). Pachytene chromosomes have been used in many studies to obtain a higher mapping resolution, due to the more decondensed state when compared to mitotic metaphase chromosomes (FRANSZ et al. 1998 ). On the other hand, a mitotic idiogram, in contrast to a pachytene one, allows the estimation of the size of each chromosome in megabase pairs due to the uniform condensation of chromosomes at the mitotic metaphase stage (LAPITAN et al. 1989 ) and is therefore useful for constructing first generation physical maps.

Telomeres, centromeres, and other repetitive sequences of L. japonicus:
The weak hybridization signals obtained at terminal chromosomal locations suggest that telomeric tracts are relatively short in L. japonicus. However, hybridization of the telomeric repeat to its centromeres was observed, indicating some similarity between these two chromosome domains. Occurrence of telomere-like repeats in centromeric heterochromatin has been demonstrated previously for animals and plants (SOUTHERN 1970 ; SIMOENS et al. 1988 ; MEYNE et al. 1990 ; MALUSZYNSKA and HESLOP-HARRISON 1991 ; RICHARDS et al. 1991 ). Although the distribution of telomere-like motifs at the centromeric DNA in Lotus is still not clear, the isolation of Ljcen1, a centromeric repeat, will help to elucidate centromere structure in legumes.

From the dispersed hybridization patterns observed with some BACs, at least three types of repetitive sequences were evident: repeats clustered at centromeres, dispersed repeats enhanced at pericentromeric regions, and uniformly dispersed repeats. Similar distribution patterns were reported for repetitive sequences in other plant species. In Arabidopsis, for instance, transposon elements show mainly a centromeric or pericentromeric distribution (ARABIDOPSIS GENOME INITIATIVE 2000).

Terminal regions of L. japonicus chromosomes were apparently enriched for single copy sequences, consistent with the findings that BAC clones with unique hybridization patterns mapped predominantly toward the chromosome ends (see also ZWICK et al. 1997 ). Furthermore, when a pool of 500 cDNA clones was used as probe for FISH in L. japonicus, signals were predominantly located at terminal regions (ITO et al. 2000 ).

Correlation of chromosomal and genetic maps:
Distorted correlation between chromosome and linkage group sizes was observed in L. japonicus. Furthermore, evaluation of the ratios between physical and genetic distances along chromosomes revealed remarkable differences and provided a clear indication for regions particularly suited for map-based cloning in this particular cross (see SANDAL et al. 2002 ).

In general, higher recombination rates were observed toward the chromosome ends. This phenomenon had previously been demonstrated in wheat, barley, and tomato (TANKSLEY et al. 1992 ; GILL et al. 1996 ; KUNZEL et al. 2000 ). In Arabidopsis and rice, however, recombination hotspots seemed to be more randomly distributed and only centromeres are not subject to recombination (SCHMIDT et al. 1995 ; HARUSHIMA et al. 1998 ; ARABIDOPSIS GENOME INITIATIVE 2000; CHENG et al. 2001B ). A possible reason for this difference is the additional accumulation of repetitive sequences at proximal chromosome regions with the increase in genome size in most plant species. This is consistent with the idea of a typical plant chromosome, with genes predominantly clustered near chromosome ends (SCHMIDT and HESLOP-HARRISON 1998 ). Because L. japonicus has a small genome size, comparable to rice, it is possible that the higher recombination frequencies at distal chromosome regions observed in the species results mainly from the influence of chromosome rearrangements on recombination in the Lotus interspecific cross. More studies on legumes are necessary to clarify this issue.

Chromosome rearrangements and their influence on the L. japonicus genetic map:
In chromosomes 1 and 3, the main distortions of the genetic map may be at least partially explained by the occurrence of a paracentric and a pericentric inversion, respectively. Comparison of genetic maps of related species such as tomato and potato, or among cereals, also suggested a large number of inversions in those genomes (TANKSLEY et al. 1992 ; MOORE 1995 ). Furthermore, a translocation was recently demonstrated in L. japonicus when two ecotypes were compared (HAYASHI et al. 2001 ). Although clustering of markers was observed in chromosome 2, no morphological change was detected when L. filicaulis and L. japonicus chromosomes 2 were compared. It is likely that the lack of both 45S and 5S rRNA genes on the genetic map contributes to the short length of this linkage group (see SCHWEIZER et al. 1987 ).

The paracentric inversion on chromosome 1 alone cannot explain the gap originally observed between linkage groups IA and IB. From the genetic data, it seems that strong selection for L. filicaulis alleles, or against L. japonicus alleles, is taking place in this region. It is likely, however, that the inversion extended the selection from one specific gene to a much larger area, influencing the segregation of markers on a chromosomal level.

The pericentric inversion on chromosome 3, on the other hand, resulted in the typical suppression of recombination in the inverted region and consequent reduction of the linkage group length. Indeed, a cluster of markers, including Nlp2, is observed in linkage group III, the total length of which is shorter than expected (Fig 4; SANDAL et al. 2002 ). Such an inversion, which brought a subtelomeric, gene-rich region to the middle of the chromosome, significantly alters chromosome organization. More detailed comparison of single copy vs. repetitive sequences in L. filicaulis would offer the opportunity to investigate whether this presumably recent inversion triggered changes in repetitive or single copy sequences to reestablish maximal gene density on the chromosome end.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY040715 and AF390569.

	ACKNOWLEDGMENTS

We thank W. F. Grant for seed material, Jörg Fuchs for fruitful discussions and constant interest, Maria Lambrou for valuable suggestions. Anna Nasmyth for help with karyotyping, and Viktoria Nizhynska for technical assistance. We are also very grateful to Johann Greilhuber and Renate Obermayer for help with DNA measurements. A.P. was supported by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico/Brazil (200249/99-1). This work was supported by the Österreichische Nationalbank grant P7172 to D.S. and, in part, by grant S8211 from the Austrian Science Foundation.

Manuscript received January 24, 2002; Accepted for publication May 8, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.. (2000) Nature 408:796-815.[Medline]

ARUMUGANATHAN, K. and E. D. EARLE, 1991  Nuclear DNA content of some important plant species. Plant Mol. Biol. Reporter 9:208-218.

BARANYI, M. and J. GREILHUBER, 1996  Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum.. Theor. Appl. Genet. 92:297-307.

CAVALIER-SMITH, T., 1985 Preface, pp. ix–x in The Evolution of Genome Size, edited by T. CAVALIER-SMITH. John Wiley & Sons, London.

CHENG, R. I.-J. and W. F. GRANT, 1973  Species relationships in the Lotus corniculatus group as determined by karyotype and cytophotometric analyses. Can. J. Genet. Cytol. 15:101-115.

CHENG, Z., C. R. BUELL, R. A. WING, M. GU, and J. JIANG, 2001a  Toward a cytological characterization of the rice genome. Genome Res. 11:2133-2141.[Abstract/Free Full Text]

CHENG, Z., G. G. PRESTING, C. R. BUELL, R. A. WING, and J. JIANG, 2001b  High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice. Genetics 157:1749-1757.[Abstract/Free Full Text]

CYRANOSKI, D., 2001  Japanese legume project may help to fix nitrogen problem. Nature 409:272.

DOLEZEL, J., J. GREILHUBER, S. LUCRETTI, A. MEISTER, and M. A. LYSÁK et al., 1998  Plant genome size estimation by flow cytometry: inter-laboratory comparison. Ann. Bot. 82:17-26.[Abstract/Free Full Text]

DONG, F., J. SONG, S. K. NAESS, J. P. HELGESON, and C. GEBHARDT et al., 2000  Development and applications of a set of chromosome-specific cytogenetic DNA markers in potato. Theor. Appl. Genet. 101:1001-1007.

FRANSZ, P., S. ARMSTRONG, C. ALONSO-BLANCO, T. C. FISHER, and R. A. TORRES-RUIZ et al., 1998  Cytogenetics for the model system Arabidopsis thaliana. Plant J. 13:867-876.[Medline]

FRANSZ, P., S. ARMSTRONG, J. H. DE JONG, L. D. PARNELL, and C. VAN DRUNEN et al., 2000  Integrated cytogenetics map of chromosome arm 4S of A. thaliana: structural organization of heterochromatin knob and centromere region. Cell 10:367-376.

FUCHS, J., M. KÜHNE, and I. SCHUBERT, 1998  Assignment of linkage groups to pea chromosomes after karyotyping and gene mapping by fluorescence in situ hybridization. Chromosoma 107:272-276.[Medline]

GILL, B. S., 1995 The molecular cytogenetics analysis of economically important traits in plants, pp. 47–53 in Kew Chromosome Conference IV, edited by P. E. BRANDHAM and M. D. BENNETT. Royal Botanic Gardens/Kew, London.

GILL, K. S., B. S. GILL, T. R. ENDO, and T. TAYLOR, 1996  Identification and high-density mapping of gene-rich regions in chromosome group 1 of wheat. Genetics 144:1883-1891.[Abstract]

GRANT, W. F., 1995  A chromosome atlas and interspecific-intergeneric index for Lotus and Tetragonolobus (Fabaceae). Can. J. Bot. 73:1787-1809.

GREILHUBER, J. and I. EBERT, 1994  Genome size variation in Pisum sativum.. Genome 37:646-655.

GREILHUBER, J. and R. OBERMAYER, 1997  Genome size and maturity group in Glycine max (soybean). Heredity 78:547-551.

HANDBERG, K. and J. STOUGAARD, 1992  Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J. 2:487-496.

HARUSHIMA, Y., M. YANO, A. SHOMURA, M. SATO, and T. SHIMANO et al., 1998  A high-density rice genetic linkage map with 2275 markers using a single F₂ population. Genetics 148:479-494.[Abstract/Free Full Text]

HAYASHI, M., A. MIYAHARA, S. SATO, T. KATO, and M. YOSHIKAWA et al., 2001  Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population. DNA Res. 8:301-310.[Abstract]

HESLOP-HARRISON, J. S., T. SCHWAZARCHER, K. ANAMTHAWAT-JÓNSSON, A. R. LEITCH, and M. SHI et al., 1991  In situ hybridization with automated chromosome denaturation. Technique 3:109-115.

HESLOP-HARRISON, J. S., G. E. HARRISON, and I. J. LEITCH, 1992  Reprobing of DNA:DNA in situ hybridization preparations. Trends Genet. 8:372-373.[Medline]

IJDO, J. W., R. A. WELLS, A. BALDINI, and S. T. REEDERS, 1991  Improved telomere detection using a telomere repeat probe (TTAGGG)_n generated by PCR. Nucleic Acids Res. 19:4780.[Free Full Text]

ITO, M., J. MIYAMOTO, Y. MORI, S. FUJIMOTO, and T. UCHIUMI et al., 2000  Genome and chromosome dimensions of Lotus japonicus.. J. Plant Res. 113:435-442.

JIANG, J., B. S. GILL, G.-L. WANG, P. C. RONALD, and D. C. WARD, 1995  Metaphase and interphase fluorescence in situ hybridization of the rice genome with bacterial artificial chromosomes. Proc. Natl. Acad. Sci. USA 92:4487-4491.[Abstract/Free Full Text]

KOUCHI, H. and S. HATA, 1993  Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Mol. Gen. Genet. 238:106-119.[Medline]

KULIKOVA, O., G. GUALTIERI, R. GEURTS, D.-J. KIM, and D. COOK et al., 2001  Integration of the FISH pachytene and genetic maps of Medicago truncatula. Plant J. 27:49-58.[Medline]

KÜNZEL, G., L. KORZUN, and A. MEISTER, 2000  Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints. Genetics 154:397-412.[Abstract/Free Full Text]

LAPITAN, N. L. V., M. W. GANAL, and S. D. TANKSLEY, 1989  Somatic chromosome karyotype of tomato based on in situ hybridization of the TGRI satellite repeat. Genome 32:992-998.

MALUSZYNSKA, J. and J. S. HESLOP-HARRISON, 1991  Localization of tandemly repeated DNA sequences in Arabidopsis thaliana.. Plant J. 1:159-166.

MEYNE, J., R. J. BAKER, H. H. HOBART, T. C. HSU, and O. A. RYDER et al., 1990  Distribution of non-telomeric sites of the (TTAGGG)_n telomeric sequence in vertebrate chromosomes. Chromosoma 99:3-10.[Medline]

MOORE, G., 1995  Cereal genome evolution: pastoral pursuits with ‘Lego’ genomes. Curr. Opin. Genet. Dev. 5:717-724.[Medline]

PEDROSA, A., M. F. JANTSCH, E. A. MOSCONE, P. F. AMBROS, and D. SCHWEIZER, 2001  Characterisation of pericentromeric and sticky intercalary heterochromatin in Ornithogalum longibracteatum (Hyacinthaceae). Chromosoma 110:203-213.[Medline]

RICHARDS, E. J., H. M. GOODMAN, and F. M. AUSUBEL, 1991  The centromere region of Arabidopsis thaliana chromosome 1 contains telomere-similar sequences. Nucleic Acids Res. 19:3351-3357.[Abstract/Free Full Text]

SANDAL, N., L. KRUSELL, S. RADUTOIU, M. OLBRYT, and A. PEDROSA et al., 2002  A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci. Genetics 161:1673-1683.[Abstract/Free Full Text]

SATO, S., T. KANEKO, Y. NAKAMURA, E. ASAMIZU, and T. KATO et al., 2001  Structural analysis of a Lotus japonicus genome. I. Sequence features and mapping of fifty-six TAC clones which cover the 5.4 Mbp regions of the genome. DNA Res. 8:311-318.[Abstract]

SCHAUSER, L., K. HANDBERG, N. SANDAL, J. STILLER, and T. THYKJAER et al., 1998  Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus.. Mol. Gen. Genet. 259:414-423.[Medline]

SCHERES, B., F. VAN ENGELEN, E. VAN DER KNAAP, C. VAN DE WIEL, and A. VAN KAMMEN et al., 1990  Sequential induction of nodulin gene expression in the developing pea nodule. Plant Cell 2:687-700.[Abstract/Free Full Text]

SCHMIDT, R., J. WEST, K. LOVE, Z. LENEHAN, and C. LISTER et al., 1995  Physical map and organization of Arabidopsis thaliana chromosome 4. Science 270:480-483.[Abstract/Free Full Text]

SCHMIDT, T. and J. S. HESLOP-HARRISON, 1998  Genomes, genes and junk: the large-scale organization of plant chromosomes. Trends Plant Sci. 3:195-199.

SCHWARZACHER, T., 1994  Mapping in plants: progress and prospects. Curr. Opin. Genet. Dev. 4:868-874.[Medline]

SCHWARZACHER, T., P. AMBROS, and D. SCHWEIZER, 1980  Application of Giemsa banding to orchid karyotype analysis. Plant Syst. Evol. 134:293-297.

SCHWEIZER, D., P. AMBROS, P. GRÜNDLER, and F. VARGA, 1987  Attempts to relate cytological and molecular chromosome data of Arabidopsis thaliana to its genetic linkage map. Arabidopsis Inf. Serv. 25:27-34.

SIMOENS, C. R., J. GIELEN, M. VAN MONTAGU, and D. INZÉ, 1988  Characterization of highly repetitive sequences of Arabidopsis thaliana.. Nucleic Acids Res. 16:6753-6766.[Abstract/Free Full Text]

SOUTHERN, E. M., 1970  Base sequence and evolution of guinea-pig -satellite DNA. Nature 227:794-798.[Medline]

STOUGAARD, J. and P. R. BEUSELINCK, 1996  Registration of Gifu B-129–S9 Lotus japonicus germplasm. Crop Sci. 36:476.[Free Full Text]

SZCZYGLOWSKI, K., D. HAMBURGER, P. KAPRANOV, and F. J. DE BRUIJN, 1997  Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes. Plant Physiol. 114:1335-1346.[Abstract]

TANKSLEY, S. D., M. W. GANAL, J. P. PRINCE, M. C. DE VICENTE, and M. W. BONIERBALE et al., 1992  High density molecular linkage maps of the tomato and potato genomes. Genetics 132:1141-1160.[Abstract]

WANZENBÖCK, E.-M., C. SCHÖFER, D. SCHWEIZER, and A. BACHMAIR, 1997  Ribosomal transcription units integrated via T-DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana.. Plant J. 11:1007-1016.[Medline]

WEGEL, E., L. SCHAUSER, N. SANDAL, J. STOUGAARD, and M. PARNISKE, 1998  Mycorrhiza mutants of Lotus japonicus define genetically independent steps during symbiotic infection. Mol. Plant-Microbe Interact. 11:933-936.

ZHONG, X.-B., J. BODEAU, P. F. FRANSZ, V. M. WILLIAMSON, and A. VAN KAMMEN et al., 1999  FISH to meiotic pachytene chromosomes of tomato locates the root-knot nematode resistance gene Mi-1 and the acid phosphatase gene Asp-1 near the junction of euchromatin and pericentromeric heterochromatin of chromosome arms 6S and 6L, respectively. Theor. Appl. Genet. 98:365-370.

ZWICK, M. S., R. E. HANSON, T. D. MCKNIGHT, M. N. ISLAM-FARIDI, and D. M. STELLY et al., 1997  A rapid procedure for isolation of C₀t-1 DNA from plants. Genome 40:138-142.

ZWICK, M. S., M. N. ISLAM-FARIDI, D. G. CZESCHIN, JR., R. A. WING, and G. E. HART et al., 1998  Physical mapping of the liguleless linkage group in Sorgum bicolor using rice RFLP-selected sorghum BACs. Genetics 148:1983-1992.[Abstract/Free Full Text]

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