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AGRICULTURAL SCIENCES
Estimating genome conservation between crop and model legume species

Hong-Kyu Choi  , Jeong-Hwan Mun  , Dong-Jin Kim  , Hongyan Zhu , Jong-Min Baek , Joanne Mudge ^¶, Bruce Roe ^||, Noel Ellis , Jeff Doyle , Gyorgy B. Kiss ^, , Nevin D. Young ^¶, and Douglas R. Cook ^, , ¶¶

Department of Plant Pathology and College of Agricultural and Environmental Sciences Genomics Facility, University of California, One Shields Avenue, Davis, CA 95616; ^¶Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108; ^||Advanced Center for Genome Technology, University of Oklahoma, Norman, OK 73019; John Innes Centre, Norwich NR4 7UH, United Kingdom; Department of Plant Biology, Cornell University, Ithaca, NY 14853; and Biological Research Center, Institute of Genetics, H-6701 Szeged, Hungary

Edited by Susan R. Wessler, University of Georgia, Athens, GA and approved August 13, 2004 (received for review March 30, 2004)

	Abstract

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Legumes are simultaneously one of the largest families of crop plants and a cornerstone in the biological nitrogen cycle. We combined molecular and phylogenetic analyses to evaluate genome conservation both within and between the two major clades of crop legumes. Genetic mapping of orthologous genes identifies broad conservation of genome macrostructure, especially within the galegoid legumes, while also highlighting inferred chromosomal rearrangements that may underlie the variation in chromosome number between these species. As a complement to comparative genetic mapping, we compared sequenced regions of the model legume Medicago truncatula with those of the diploid Lotus japonicus and the polyploid Glycine max. High conservation was observed between the genomes of M. truncatula and L. japonicus, whereas lower levels of conservation were evident between M. truncatula and G. max. In all cases, conserved genome microstructure was punctuated by significant structural divergence, including frequent insertion/deletion of individual genes or groups of genes and lineage-specific expansion/contraction of gene families. These results suggest that comparative mapping may have considerable utility for basic and applied research in the legumes, although its predictive value is likely to be tempered by phylogenetic distance and genome duplication.

The 20,000 legume species are divided into three subfamilies: Mimosoideae, Caesalpinioideae, and the numerically and economically dominant Papilionoideae (2). With the notable exception of peanut, the important crop legumes occur in two Papilionoid clades, referred to here as the "phaseoloid" and "galegoid" legumes (Table 1). Despite their close phylogenetic affiliations (Fig. 1), the genetic systems represented within this group are diverse, ranging from simple autogamous diploids to complex out-crossing polyploids. Genome size also varies widely among legumes, with pea having a genome size 10 times that of some related diploid genera.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Taxonomic relationships within the two major clades of crop legumes, the prevailing view of phylogeny for the species under analysis, with divergence times estimated based on Penalised Likelihood analysis (2). Most crop legumes occur either within the galegoid clade, including tribes Viceae, Trifolieae, and Cicereae, or within the phaseoloid clade, which is synonymous with the tribe Phaseoleae. MYA, million years ago.

A pressing need in legume genomics is to integrate knowledge gained from the study of model legume genomes with the biological and agronomic questions of importance in the crop species. Comparative genetic mapping is well established in several plant families, most notably the Poaceae (4), where initial studies predicted that synteny would greatly facilitate gene discovery among related species (5, 6). However, even closely related grass species (7, 8), in some cases members of the same species (9), can exhibit significant divergence in genome organization. It is important to know whether similar features are prevalent in other plant families, in particular because the extent of such differences may define the limits of comparative structural genomics as a strategy for applied agriculture.

Here we combined genetic and phylogenetic analyses to map putatively orthologous genes across seven legume species. Complementing the genetic linkage analysis, we surveyed the conservation of genome microstructure between M. truncatula and L. japonicus and M. truncatula and Glycine max (soybean) by comparing fully sequenced bacterial artificial chromosome (BAC) clones. The combined genetic, phylogenetic, and genomic analyses demonstrate extensive conservation of gene order and orthology between the crop and model legumes and also identify features of structural divergence between these genomes.

	Materials and Methods

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Plant Material and Mapping Populations. Plant genotypes used for genetic mapping are shown in Table 1. The core maps for each of the genomes under analysis have been described (6–15). In the cases of Pisum sativum and Vigna radiata, mapping populations were composed of highly selected genotypes chosen so that recombination break points were spaced evenly throughout the genome (16). BAC clones were mapped in M. truncatula by means of simple sequence repeat polymorphisms discovered in the course of shotgun sequencing.

Development of Cross-Species Genetic Markers. The development and genetic mapping of gene-specific markers were as described by Choi et al. (10). BLASTN [National Center for Biotechnology Information (NCBI), Bethesda] was used to identify conserved sequences between ESTs of the legume M. truncatula and other legume species. Multiple sequence alignments, with the Arabidopsis genomic sequence used to infer intron position, facilitated design of PCR primers that anneal to conserved exon sequences and amplify across more diverged introns. Polymorphisms (Table 2, which is published as supporting information on the PNAS web site) were identified by sequencing PCR products from parental lines (Table 1), followed by manual inspection of alignments and chromatograms. Markers were typically analyzed as cleavable amplified polymorphic sequences (10). Single-nucleotide polymorphisms that did not alter a restriction site were scored by DNA sequencing of PCR products. Resequencing was used to confirm or refute apparently ambiguous data.

Phylogenetic Analysis. Neighbor-joining trees were rooted by using the closest Arabidopsis sequence as an outgroup or left unrooted where no close homolog was present in Arabidopsis. Phylogenetic analyses were conducted by using parsimony options in PAUP^* (17). The principal analysis involved 100 searches with random taxon addition and tree bisection–reconnection (TBR) branch swapping, with maxtrees set to increase without limit. Support for branches was assayed by 100 bootstrap replicate searches using simple taxon addition, TBR branch swapping, and maxtrees set to 1,000.

Microsynteny Analysis. Accession numbers for sequenced BACs are given in Tables 2–5, which are published as supporting information on the PNAS web site. Homologous transformation-competent BAC (TAC) clones of L. japonicus were obtained from NCBI (18). Ab initio gene prediction involved the eudicot version of FGENESH (www.softberry.com/berry.phtml?topic=gfind). Gene prediction based on identity to transcribed sequences was obtained by BLASTN against The Institute for Genomic Research M. truncatula or L. japonicus Gene Index databases (www.tigr.org/tdb/tgi). Additional predicted proteins were identified by means of BLASTX (NCBI) against the NCBI nonredundant protein database. BLASTP (NCBI) was used to compare predicted proteins between M. truncatula and L. japonicus clone pairs, with a maximum E value cutoff of e^-10 and a median E value of <e^-100 for 533 protein pairs. REPEATMASKER (http://repeatmasker.genome.washington.edu/cgi-bin/Repeat-Masker) was used to screen interspersed repeats, transfer RNA, and low-complexity DNA sequences.

	Results

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Development of Cross-Species Gene-Specific Genetic Markers. We sought to develop cross-species genetic markers where locus orthology was an explicit aspect of the analysis. One hundred sixty-seven gene-specific PCR primer pairs were tested for amplification and polymorphism by using the parents of available mapping populations in M. truncatula, Medicago sativa, Pi. sativum, L. japonicus, V. radiata, Phaseolus vulgaris, and G. max (Table 1). To test the orthology of these genetic markers, we constructed phylogenies for 24 of the markers that produced high-quality sequence data for at least four of the six species under analysis (excluding M. sativa) (as shown by example in Fig. 6, which is published as supporting information on the PNAS web site). Combining alignment matrices and analyzing the data by neighbor-joining and maximum parsimony methods yielded a tree depicting genomic relationships. The monophyly of the galeoid and phaseoloid clades was strongly supported, as was the sister relationship of Phaseolus and Vigna. Analysis of individual gene trees supports the Loteae as sister to the galegoid clade. As a further test of sequence orthology, 11 markers with unambiguous global alignments were analyzed across 95 diploid legume species (Fig. 7, which is published as supporting information on the PNAS web site) spanning the diversity of the Fabaceae. The overall agreement with published phylogenies of the family (2, 19) supports the orthology of these genes and the utility of these exon-derived markers as tools for comparing legume genomes.

Macrosynteny Analysis Among M. truncatula, M. sativa, Pi. sativum, G. max, V. radiata, and Ph. vulgaris. For purposes of establishing a comparative genetic map spanning galegoid and phaseoloid species, we analyzed marker segregation in M. truncatula, M. sativa, Pi. sativum, and V. radiata. In addition to the markers developed based on phylogenetic criteria, we analyzed 60 primer pairs developed based on homology to genetic markers in G. max and 117 additional markers developed for the M. truncatula core genetic map (10). In all cases, M. truncatula was the central point of comparison. Comparisons between the two Medicago species and between Ph. vulgaris and V. radiata have been presented elsewhere (11, 12) and are included here for the sake of integration.

The pea genome is much larger (10 times) than that of M. truncatula and has a base chromosome number of 7, compared to 8 in M. truncatula. Despite these overt differences, analysis of 57 gene-specific markers reveals broad conservation of genome structure, with the major evident differences being sites of inferred chromosomal rearrangements. An average of eight colinear genetic markers were present for each pea chromosome, and in only one case (i.e., marker PTSB) did we identify the possible translocation of an orthologous gene. Instead, we identified a limited number of chromosomal translocation events such as that shown for the top terminal region of PsLGIII and the bottom portion of MtLG2 (Fig. 2). MtLG6 could not be effectively integrated into the Pi. sativum genetic map, due to a lack of comparative markers in this linkage group. This result corresponds with the previous observation that MtLG6 is rich in heterochromatic DNA (20) and relatively poor in transcribed genes (10). The absence of a corresponding single linkage group in pea suggests that chromosomal rearrangements involving M. truncatula chromosome 6 might be responsible for the difference in chromosome number between these two species.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Macrosyntenic relationships among legumes with reference to M. truncatula linkage groups 2 and 3. Mt, M. truncatula; Ms, M. sativa; Ps, Pi. sativum; Vr, V. radiata; Gm, G. max; Pv, Ph. vulgaris. Shared comparative markers are denoted by bold lettering. Extrapolated marker positions are denoted by dotted semicircle lines and are drawn only when the colinearity is conserved in the neighboring regions. The comparison between V. radiata and Ph. vulgaris is based on previous work (11–15).

Soybean is also a member of the phaseoloid clade but has a polyploid genome that is predicted to have undergone duplication since its divergence from other Phaseoleae. Sixty loci were mapped in common between M. truncatula and soybean, with the majority of markers derived from homologs of soybean restriction fragment length polymorphism probes identified in M. truncatula (10). A complicating feature of cultivated soybean is a low level of polymorphism, as evidenced in Table 1, which when taken together with the high level of soybean gene duplication significantly reduced our ability to interpret the comparative map between these two species. Nevertheless, 38% of the markers revealed putative synteny between M. truncatula and soybean, identifying 11 colinear blocks between the two genomes (Figs. 2 and 8). Yan et al. (21, 22) report genome-wide conserved microsynteny between the genomes of M. truncatula and soybean, with 54% of 50 soybean contig groups showing conserved microsynteny to M. truncatula. Five of the extensively microsyntenic contigs (21) were mapped in M. truncatula in this study, three of which, namely A095, A064, and A315, were mapped in regions showing putative synteny between M. truncatula and soybean.

Microsynteny Among Papilionoid Legume Genomes. To determine the extent to which macrosytenic relationships identified by genetic mapping are indicative of conserved genome microstructure, we compared putatively orthologous large insert clone pairs [i.e., BAC or transformation-competent BAC (TAC) clones] between M. truncatula and L. japonicus and between M. truncatula and soybean. The Loteae are a sister group to the galegoid legumes (Fig. 1), and, thus, L. japonicus has a more recent ancestry to M. truncatula than to soybean. Sixty-three sequenced BAC and TAC clone pairs containing an average of nine microsyntenic gene pairs were mapped between the M. truncatula and L. japonicus genomes. As shown in Fig. 3, the genomes are highly syntenic, with macrosynteny punctuated by rearrangements that frequently involve translocation of chromosome arms (Fig. 3), reflecting the difference of six chromosomes in L. japonicus vs. eight chromosomes in M. truncatula.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Macrosyntenic relationship of M. truncatula and L. japonicus. Sixty-three pairs of sequenced BAC clones (Table 3), representing putatively orthologous loci with known genetic map positions, were used to construct a comparative map between M. truncatula and L. japonicus. Based on the independent selection of clones in each species, many clone pairs possess only partial overlap. Line color indicates the number of conserved genes between two clones: black, two; blue, three to four; red, five or more.

The example of a 141-kb region of M. truncatula at genetic marker MtEIL is shown in Fig. 4. All 16 predicted M. truncatula genes possess strong similarity to annotated genes in the Arabidopsis genome. A remarkable feature of this region is the frequent occurrence of local gene duplication, including two argonaut-like genes (MtEIL-e1–2), two blue-copper-binding proteins (MtEIL-k1–2), five kinase-like genes (MtEIL-l1–4), and two I-box-binding factors (MtEIL-m1–2). Analysis of the corresponding 97-kb segment from L. japonicus (LjEIL) revealed region-wide colinearity with the MtEIL contig. Ten distinct genes were identified in LjEIL, with only a single case of tandem duplication (LjEIL-d1–2). All 10 L. japonicus genes and a transfer RNA^Leu had homologs in the MtEIL region. Six of the 16 distinct homologs from the MtEIL and LjEIL regions exhibit a network of microsynteny with two homeologous regions of Arabidopsis chromosomes 2 and 5, respectively (Fig. 4).

View larger version (35K): [in this window] [in a new window]   Fig. 4. Microsynteny between M. truncatula and L. japonicus. Microsynteny at the MtLG5 locus MtEIL2–1. Gene annotations are given in Table 4. Genes shown in pink correspond to genetic markers. Letters denote unique gene annotations, with numbers denoting tandem duplication.

	Discussion

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The idea that conserved genome synteny can facilitate transfer of knowledge among related species of plants is best articulated in the case of the grasses (5, 6). It is increasingly clear, however, that there are many exceptions and complexities to the "rule" of conserved grass genome synteny, because even in regions of genetically defined synteny the insertion/deletion and duplication of genes can contribute to significant divergence (e.g., refs. 7 and 8). Recent studies using the genome of Arabidopsis as a reference document a history of genome duplication in angiosperms (23–25), followed by significant erosion of local gene content. In retrospect, it appears that genome synteny is unlikely to be well conserved beyond the taxonomic level of plant families (4). As a consequence, testing the "grass model" of genome synteny for other agronomically important families is an important objective for plant sciences.

In the case of the legume family of plants, there are numerous species with a long history of traditional breeding but with limited molecular characterization, and there are several species that are characterized at both the genetic and genomic levels. Determining the extent of synteny (and the frequency and nature of its exceptions) among these legume genomes was the focus of this study. We report that synteny is high among closely related species, and that the degree of synteny declines with increasing phylogenetic distance. Although this study is unusual in its use of explicit phylogenetic measures to assess gene orthology and its incorporation of a large genome sequence data set, the features of genome conservation and divergence that we describe are typical of those observed in comparative analysis of both plant and animal genomes.

The high level of conservation between the genomes of M. truncatula and Pi. sativum is particularly striking given the 10 times larger genome (26) and one less chromosome in Pi. sativum. Much of the expansion in Pi. sativum genome size may be due to retroelements (27), but, whatever the mechanism, it has done little to disrupt macrosyntenic relationships. M. truncatula LG6 could not be effectively integrated into the Pi. sativum genetic map. MtLG6 is interesting for several reasons: (i) it is the shortest and most highly heterochromatic of the chromosomes (20), (ii) it is underrepresented for randomly selected EST markers (5), and (iii) it is remarkably rich in resistance gene analogs (RGAs) (28). The inability to establish synteny in this study between MtLG6 and Pi. sativum is undoubtedly due to the low frequency of non-RGA EST markers in MtLG6 (10) and the fact that the genetic maps of pea (13) are not well populated by RGA markers. However, parallel analyses conducted by Kaló et al. (29) suggest that M. sativa LG6 is syntenic with several regions in the pea genome, in particular PsLGVI and PsLGVII (Fig. 7d). The absence of a corresponding single linkage group in pea suggests that chromosomal fission/fusion events involving Medicago chromosome 6 might be responsible for the reduction of chromosome number in Pi. sativum.

L. japonicus (tribe Loteae) and M. truncatula represent the two best-characterized legume genomes. Although there are no important crop legumes within the Loteae, the relatively recent divergence and sister-clade relationship to the galegoid legumes (Fig. 1) offers a potentially useful point of comparison to M. truncatula. We determined that M. truncatula and L. japonicus share a remarkably high level of conserved macrosynteny, dominated by a few large chromosome arm-size rearrangements. The availability of fully sequenced large insert clones [i.e., BACs and transformation-competent BAC (TACs)] at each of these genetically syntenic loci provided an opportunity to evaluate the correlation between genetic macrosynteny and sequence microsynteny. Conserved microsynteny was characterized by 80% of close homologs in the same order and transcriptional orientation, similar to values obtained between human and mouse (30) and within the range identified for the grasses (7). The current analysis also reveals significant divergence between these two legume genomes, with the insertion or deletion of individual or groups of genes accounting for 20% divergence of gene content in microsyntenic intervals. Species-specific tandem duplication of genes accounted for an additional 12–17% divergence of gene content, and each species possessed a unique distribution of mobile DNAs. Of 21 tandemly duplicated genes, only one duplication was reciprocal. Similarly, of 26 cases of mobile DNAs, only one mobile DNA was potentially syntenic. The lack of ancestral tandem duplication is suggestive of either efficient removal of tandem duplicates that predate speciation or a recent increase in the rate of tandem duplication. Moreover, the observation that tandemly duplicated genes are occasionally interspersed with single copy genes (Table 4) suggests that purification of duplicates by homologous recombination would simultaneously eliminate the intervening single copy gene(s). Such a mechanism could explain, at least in part, the loss of gene homologs from microsyntenic regions.

Syntenic relationships were significantly more convoluted between the galegoid and phaseoloid clades. Twenty-five percent of genetically mapped orthologous genes were potentially nonsyntenic, resulting in smaller regions of colinearity than those observed between M. truncatula and Pi. sativum or between M. truncatula and L. japonicus. This fragmentation of synteny might be expected based solely on the differences in chromosome number between these two clades. Despite the relatively large number of genetic markers used for comparison, synteny between M. truncatula and soybean was difficult to characterize. We attribute this situation to a combination of recent duplication and low rates of polymorphism in the soybean genome. Nevertheless, comparison of putatively orthologous BAC clone pairs revealed significant conservation of microsynteny between M. truncatula and soybean, consistent with previous comparison of other genome regions between these two species (22, 31). In all cases, conservation of microsynteny was significantly greater between legume genomes than between legume genomes and the corresponding regions of the Arabidopsis genome (Figs. 4 and 9).

The genetic mapping of orthologous genes across multiple species provides an opportunity to propose an integrated view of legume synteny. As shown in Fig. 5, the results suggest broad macrosynteny, particularly within the galegoid or phaseoloid legumes, punctuated by chromosomal rearrangements that increase in frequency with phylogenetic distance. The inclusion of phylogenetic measures in marker analysis also aided the inference of genomic rearrangements. For example, in M. truncatula, orthologous marker PTSB maps to LG5, which is highly conserved with PsLGI (Fig. 2). In Pi. sativum, PTSB maps to a nonsyntenic region of PsLGIII, near the point of an inferred chromosomal rearrangement. The combination of PTSB orthology to a marker on MtLG5 and disrupted synteny of the flanking markers are consistent with a complex genome rearrangement involving both chromosomal fragment translocation and single gene translocation. Such events are likely to be significantly more frequent between the galegoid and phaseoloid legumes, because several markers that map to conserved regions in the galegoid legumes occur in nonsyntenic regions of V. radiata (Figs. 2, 5, and 8). This result is consistent with significant genomic changes that must underlie differences in chromosome number between galegoid (typically eight chromosomes) and phaseoloid (typically 11 chromosomes) lineages.

View larger version (71K): [in this window] [in a new window]   Fig. 5. Consensus comparative map data for six legume species. Species abbreviations are as in Fig. 2. S and L denote short and long arms of each chromosome in M. truncatula (20). Synteny blocks are drawn to scale based on genetic distance. Solid lines, postulated rearrangements; double-headed arrows, postulated inversions. Gene-specific markers that disrupt synteny are S-SHMT, R-RNAH, T-TRPT, M-MMK1, P-PTSB, A-ARG10, D-6DCS, and E-REP.

	Conclusion

Top Abstract Materials and Methods Results Discussion Conclusion References

Although the current study documents substantial conservation between the genomes of crop and model legumes, it also reveals features of genome divergence. The degree to which genome synteny can facilitate cross-species analysis of gene function will depend both on the conservation of gene order and content, as well as on the frequency with which similar traits have a common genetic basis in different species. An indication that this latter criterion might not always be met is suggested by a recent study of branching in foxtail millet (34). By contrast, similar studies of symbiotic nitrogen fixation in legumes (reviewed in ref. 3) demonstrate that functionally conserved genes occupy syntenic positions across the diversity of legume species analyzed in this study. Moreover, even large and rapidly evolving gene families, such as the nucleotide-binding site-leucine-rich repeat resistance gene homologs, can occupy ancestral genome locations among legumes (28). Testing the extent to which inferences made from comparative genomics can be translated to practical applications in crop improvement represents one of the major current challenges facing plant biology.

	Acknowledgements

 
This work was supported by National Science Foundation Grant 0110206 (to D.R.C., N.D.Y., and D.-J.K.); the Samuel Roberts Noble Foundation (to B.R.); the European Union MEDICAGO project (QLG2-CT-2000-30676) and the Biotechnology and Biological Sciences Research Council (to N.E.); and Nemzeti Kutatás es Feilesztesi Program and the (HNDRP) [Országos Tudományos Kutatási Alap Programok (OTKA) and T038211, to G.B.K.].

	Footnotes

 
This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: BAC, bacterial artificial chromosome; NCBI, National Center for Biotechnology Information.

H.-K.C., J.-H.M., and D.-J.K. contributed equally to this work.

^¶¶ To whom correspondence should be addressed. E-mail: drcook{at}ucdavis.edu.

© 2004 by The National Academy of Sciences of the USA

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