Synteny of Prunus and other model plant species

Background Fragmentary conservation of synteny has been reported between map-anchored Prunus sequences and Arabidopsis. With the availability of genome sequence for fellow rosid I members Populus and Medicago, we analyzed the synteny between Prunus and the three model genomes. Eight Prunus BAC sequences and map-anchored Prunus sequences were used in the comparison. Results We found a well conserved synteny across the Prunus species – peach, plum, and apricot – and Populus using a set of homologous Prunus BACs. Conversely, we could not detect any synteny with Arabidopsis in this region. Other peach BACs also showed extensive synteny with Populus. The syntenic regions detected were up to 477 kb in Populus. Two syntenic regions between Arabidopsis and these BACs were much shorter, around 10 kb. We also found syntenic regions that are conserved between the Prunus BACs and Medicago. The array of synteny corresponded with the proposed whole genome duplication events in Populus and Medicago. Using map-anchored Prunus sequences, we detected many syntenic blocks with several gene pairs between Prunus and Populus or Arabidopsis. We observed a more complex network of synteny between Prunus-Arabidopsis, indicative of multiple genome duplication and subsequence gene loss in Arabidopsis. Conclusion Our result shows the striking microsynteny between the Prunus BACs and the genome of Populus and Medicago. In macrosynteny analysis, more distinct Prunus regions were syntenic to Populus than to Arabidopsis.

nuts like almond, and lumber trees like black cherry. Other important fruit producing crops in Rosaceae include apple (Malus), pear (Pyrus), raspberry/blackberry (Rubus) and strawberry (Fragaria). In addition, Rosaceae contains a wide variety of ornamental plants including roses, flowering cherry, crabapple and quince.
Significant conservation of the genomes among the Prunus member species has been shown by comparative mapping studies [3]. For example, comparisons of the anchor marker positions on the Prunus reference map with those on 13 other maps showed that the genomes of the diploid Prunus species are essentially collinear [3]. Large collinear blocks among different genera in Rosaceae, such as Prunus and Malus, were also detected [3]. Similar conserved collinearity of shared markers has also been observed among other closely related species within the grasses [4], legumes [5,6] and between potato and tomato in the Solanaceae [7].
With the increase in available genomic sequence data, more laboratories have looked at collinearity at the genome sequence level. The synteny between rice and other cereals are shown to be high [8], and substantial micro-collinearity among legumes was reported [9][10][11]. These comparisons have revealed that the conserved syntenic regions, detected by marker collinearity, were often interrupted by small genome rearrangements such as insertions, deletions, inversions and translocations [12][13][14]. Large scale sequence data has also enabled the detection of micro-collinear regions in less closely related species without apparent macrosynteny [15]. Small scale microsynteny was observed when a 276 kb region of the model legume Medicago (rosid I) was compared with the genomic sequence of Arabidopsis (rosid II) [16]. Conversely, no macrosynteny was found when the two genomes were compared using 172 mapped markers of Medicago [16]. Significant levels of local synteny were also detected covering segments of 1 Mb of Arabidopsis and regions of <5 cM in lettuce and sunflower, members of the asterids, even though the macrosyntenic patterns covering larger segments were not evident [17]. A recent study detected microsynteny between coffee, another asterid species, and the Arabidopsis genome using putative orthologous sequences and BAC ends [18]. We have previously detected conserved syntenic regions between Prunus and Arabidopsis using 475 peach ESTs anchored to Prunus genetic maps and 1097 peach ESTs anchored to BAC contigs [17]. The conserved syntenic regions were short and fragmentary, and often the Prunus regions matched to more than one Arabidopsis block. This complex network of microsynteny, often including non-collinear regions, between Arabidopsis and the distantly related species suggests multiple genome duplications followed by differential gene loss [15][16][17][18][19][20][21]. Large segmental or whole genome duplication followed by gene loss seems to be prevalent in the evolution of all flowering plant genomes, and the degree of genome instability seems to be higher in some species of Brassicaceae, such as Arabidopsis, and in some species of Poaceae as well [22][23][24].
Synteny analysis across species provides insight on the evolutionary relationships between different lineages and also the opportunity to study the relationship between genome structure and function of organisms. The microcollinear regions conserved between model organisms like Arabidopsis and lesser-studied organisms can also facilitate marker saturation and candidate gene searches. For example, micro-collinearity data between Arabidopsis and rice has been utilized in the improvement of phylogenetic resolution of the expansin gene family [25]. Multiple rounds of polyploidization or large segmental duplication followed by gene loss, however, can greatly obscure the synteny, impeding the transfer of genomic knowledge from model species genomes to those of less well characterized species.
The recent availability of the whole genome sequence of Populus trichocarpa provides another resource in the detection of conserved synteny among plant genomes [26]. Also available are the partially sequenced Medicago genome [11]. Populus and Medicago belong to rosid I which also includes Prunus; hence Prunus is evolutionarily closer to them than Arabidopsis which belongs to rosid II. Populus is of particular interest to compare with Prunus since Populus is completely sequenced, is a fellow tree species and it appears to be more stable compared to the Arabidopsis genome; nucleotide substitution, tandem gene duplication and gross chromosomal rearrangement proceed more slowly in Populus then in Arabidopsis [26]. The detection of melon BAC regions that are more conserved in Populus than in Medicago or Arabidopsis [27] and the report of papaya BACs that are more collinear in Populus than in Arabidopsis [28] also suggest that the Populus genome may be useful in the exploration of distantly related species genomes.
With near completion of the peach physical map [29] and the development of EST-derived genetic markers, the numbers of sequences that are anchored to Prunus genetic maps or the peach physical map have doubled since our previous synteny analysis between Prunus and Arabidopsis. Also available to us are four homologous Prunus BAC sequences and four additional peach BAC sequences. Using these data, we analyzed the degree and the pattern of the conserved syntenic region between Prunus and Populus or Arabidopsis. We also compared the Prunus BAC sequences with the partially sequenced Medicago genome to gain further insight on the genome evolution of the related plant species.
The predicted gene sequences in these Prunus BACs and their Populus, Medicago and Arabidopsis homologs were used to find conserved syntenic regions. Syntenic groups were identified when the distance between the two adjacent matches was less than 200 kb and when the syntenic regions contain at least four gene pairs. We first detected collinear syntenic regions using the DAGchainer program [31] and then merged the overlapping syntenic regions. This method gave us a more comprehensive view of the syntenic regions which include sections where gene contents are conserved but not the gene order due to smallscale genome rearrangements.
The Prunus BACs had syntenic regions in the Populus and Medicago genome, but no detectable syntenic regions in Arabidopsis (Figure 1, 2). Interestingly, two distinct Populus genomic regions were detected for each of the Prunus BAC regions, and the two corresponded to the duplicated Populus genomic regions that were generated by the most recent whole-genome duplication event, which occurred 60-65 million years ago [26]. Figure 1 shows these syntenic regions in LG_V and LG_VII of the Populus genome that are conserved with BACs from across the Prunus species: peach, plum, and apricot. The syntenic regions span 50 kb in the plum BAC, 44.3 kb in the peach BAC, 37.5 kb in the apricot BAC, and 60.4 kb and 117.2 kb in LG_V and LG_VII of the Populus genome, respectively. These three Prunus BACs also showed conserved synteny with Medicago, in three different chromosomal regions ( Figure 2). Detection of more than one syntenic region in Medicago correlates with the whole genome duplication event in Medicago, which was proposed to have occurred after the split between the legumes and Salicaceae (poplar) and before the separation of Medicago and Lotus [11]. Cannon et al. [11] shows extensive synteny between chr5 and chr8 of Medicago, and we detected two regions in chr5 and chr8 of Medicago that are syntenic to the Prunus BACs ( Figure  2). The gene order was well conserved in all these syntenic regions without any signs of translocation, but there was a 142 kb gap in the syntenic region in chr08 of the Medicago genome ( Figure 2). No syntenic regions in Arabidopsis were detected for these four Prunus BACs.
The two overlapping peach BACs, 028F08 and 082I18, with a total of 38 predicted genes had remarkably wellconserved syntenic regions in LG_VI and LG_XVI of the Populus genome ( Figure 3). Of the 38 predicted genes in the combined peach BACs, 26 and 19 showed conserved synteny with the regions in LG_VI and LG_XVI, respectively. In combination, 30 out of 38 predicted peach genes belong to the syntenic groups that are conserved in LG_VI or LG_XVI of the Populus genome. The gene order, as well as the gene content, was conserved between Populus and peach. The syntenic regions span 133 kb in the peach BACs, and 395 kb in LG_VI and 477 kb in LG_XVI of the Populus genome. The actual sizes of the syntenic regions can be larger since the synteny extends to the end(s) of the BAC clone ( Figure 3). There were signs of tandem gene duplication in both the Populus region and the Prunus region ( Figure 3). In comparison, two small syntenic regions with only four or five genes spanning 49 kb or 27 kb of the peach BACs and 13 kb or 7.3 kb of the Arabidopsis genome were detected in Arabidopsis ( Figure 3). The overlapping peach BACs also detected a syntenic region in the Medicago genome ( Figure 4). Two parts in the combined peach BAC detected different regions in the chr02 of Medicago, separated by 7.3 Mb, suggesting a translocation event or potential miss-assembly due to the incompleteness of the Medicago genome assembly (Figure 4).
The peach BAC PpN089G02 detected three conserved syntenic regions in Populus LG_II and LG_VII ( Figure 5A). The block in LG_II and a block in LG_VII shared four gene matches to the BAC PpN089G02, and the other region in LG_VII were syntenic to an overlapping region (Figure 5A), suggesting all these three blocks represent a duplicated region that went through selective gene loss. The syntenic regions span 112 kb in the peach BAC PpN089G02, and around 60 kb in all three Populus regions. The peach BAC pPn31C7 showed conserved syntenic regions in LG_II and LG_V of the Populus genome, which share five gene pairs ( Figure 5B). The syntenic regions span 35 kb in the peach BAC pPn31C7, and 48 kb and 53 kb of LG_II and LG_V of the Populus genome. In the analysis with the partially sequenced Medicago genome, we found one syntenic region that shares four genes with the peach BAC PpN089G02 ( Figure 5A). No syntenic regions for the two peach BACs, PpN089G02 and pPn31C7, were detected in the Arabidopsis genome.
Conserved syntenic regions between the two overlapping peach BACs and the Poplulus or Arabidopsis genomes Figure 3 Conserved syntenic regions between the two overlapping peach BACs and the Poplulus or Arabidopsis genomes. When peach sequences match more than one genes in Populus, the matches with lower E value (lower PID when the E value is the same) are depicted with dotted lines. The matches between the peach sequences and the Arabidopsis genes are depicted with dotted lines. Only the predicted genes showing homology with genes in other genomes, not the intervening genes, are depicted. The numbers on the left side of the bar stand for base pair positions in the Prunus BACs, the Populus linkage groups, or the Arabidopsis genome. The lengths of the syntenic regions and the total numbers of predicted genes in the regions are given below the bar.
Conserved syntenic regions between the two overlapping peach BACs and the Medicago genome The fact that we detected microsynteny between all the Prunus BACs and Populus genome is noteworthy. Also the level of conserved synteny within the individual blocks (synteny quality) between the Prunus BACs and Populus was strikingly high considering the divergence between these two genomes 99 million years ago [32]. Cannon et al [11] has reported that the quality of all predicted orthologous blocks between the legumes Medicago truncatula and Lotus japonicus that diverged 40 million years ago, is 54% ± 14%. They calculated "synteny quality" as twice the number of matches divided by the total number of genes in both segments after excluding transposable elements and collapsing tandem duplications. The syntenic quality between Prunus BACs and the Populus genome ranged from 20 to 44%, without considering the orthology of the blocks or collapsing tandem duplications (Table 1). We only used the syntenic regions where the synteny did not extend to the border of the BAC to calculate the syntenic quality. The syntenic quality between the Prunus BACs and Medicago genome ranged from 11 to 40% (Table 1).
The E values of most of gene matches in the syntenic regions were considerably lower than our cut-off value, 1E-6 (see Methods

Synteny Analysis between map anchored peach sequences and the Populus or the Arabidopsis genome
To assess the degree of macrosynteny conservation, we used two sets of map-anchored Prunus sequences in the analysis. One set comprised of 1093 sequences that are anchored to the TxE Prunus reference map [33]. The majority of these sequences are peach fruit ESTs but also include sequences from markers that were directly used in mapping. Some ESTs are anchored to multiple positions and the number of anchored sequences in each linkage group is as follows: 281 in G1, 364 in G2, 268 in G3, 424 in G4, 221 in G5, 209 in G6, 65 in G7 and 310 in G8. Another set comprised of peach EST sequences that are anchored to the peach physical map. This data comprised of 2140 EST sequences that are anchored to 1500 BAC contigs and their Populus and Arabidopsis homologs.
These map-anchored sequences and their Populus and Arabidopsis homologs were used to find conserved syntenic regions. The syntenic groups were selected when the distance between the two adjacent matches were less than the maximum distance and when the syntenic regions contain at least four gene pairs (See Methods).
There were 8 and 17 syntenic regions containing four or more gene pairs between Prunus TxE map and Populus or Arabidopsis. Between the peach physical map and Populus or Arabidopsis, 10 and 17 syntenic regions were detected, respectively. Some syntenic groups between Prunus and Arabidopsis were not collinear, but all the groups between the Prunus and Populus were collinear (Table 2). In the synteny analysis with the TxE genetic map anchored sequences, the number of gene pairs were similar: four to five in Populus and four to seven in Arabidopsis genome ( Table 2). The syntenic groups span 5 to 15.7 cM in both cases, but span longer in Populus (424 kb -3.26 Mb) than in Arabidopsis (197 kb -1 Mb), reflecting the larger intergenic space in Populus than in Arabidopsis (see Methods). Similarly, the peach physical map anchored sequences detected longer syntenic regions in Populus (158 kb -1.1 Mb) than in Arabidopsis (42 -812 kb), even though the length of the syntenic regions in the physical map was similar in both cases ( Table 2). All ten syntenic groups between the peach and Populus had four gene pairs, but eight out of 17 syntenic groups between the peach and Arabidopsis had more than four gene pairs ( Table 2). As seen in the analysis with the Prunus BACs, the E values of most of the gene matches in the syntenic regions were considerably lower than our cut-off value, 1E-6 (see Methods). The median E values were between 2E-39 and 3E-51 (Table 2), and 90% to 100%, depending on the data sets, of the gene matches had the E value of less than 1E-10.
Interestingly, despite the smaller number of syntenic groups in Populus than in Arabidopsis, the syntenic groups in Populus actually detected the similar or more distinct regions in the Prunus TxE map and physical map, respectively (Table 3). Table 3A shows that the 17 syntenic groups in Arabidopsis and the eight syntenic groups in Populus were matched to similar regions in three linkage groups of the TxE genetic map, G3, G5 and G8. In other words, the same regions in the TxE map detected more than twice the number of syntenic regions in Arabidopsis than in Populus, suggestive of multiple genome duplica- Only the blocks that the synteny does not extend to the border of the BAC were used. * Twice the number of matches divided by the total number of genes in both segments  1 Range of the lengths of the syntenic blocks. 2 Numbers in parenthesis are the approximate lengths in peach genome, calculated from the TxE map size 524 cM [39] and the peach genome size 290 Mb [43]. 3 One unit length of the BAC contig corresponds to about 1.25 kb (See Methods). tion events in Arabidopsis. An example is shown in Figure  6; a region in G3 of the TxE map that displays synteny with four distinct regions in the Populus genome and seven distinct regions in the Arabidopsis genome. In the analysis with peach physical map anchored sequences, ten groups in Populus matched to five different BAC contigs, but 17 groups in Arabidopsis matched to only three different BAC contigs (Table 3B). Five and 14 of the syntenic groups were matched to one BAC contig, ctg259 (Table 3B). Even though the BAC contig ctg259 had almost three times more syntenic regions in Arabidopsis than in Populus, the ctg259 regions that are syntenic to Arabidopsis were only 18% larger than those in Populus (Table 3B).
We also analyzed for Prunus blocks that match to more than one site in the Populus or Arabidopsis genome. These were detected by selecting syntenic regions that share more than three Prunus sequences. We found one Prunus block in G5 of the TxE map matching to regions in two different Populus linkage groups, LG_II and LG_XIV (Figure 7A). We also found that a Prunus block in ctg468 of the physical map matches to regions in LG_II and LG_V ( Figure 7B). Tuskan et al [26] have reported that major part of the LG_II is homologous to either LG_V or LG_XIV, generated by the most recent shared wholegenome duplication event. The blocks that we detected in Prunus map G5 and peach BAC contig ctg468 showed syn-Syntenic groups covering the three trait loci anchored to G3 of the Prunus TxE Figure 6 Syntenic groups covering the three trait loci anchored to G3 of the Prunus TxE. Map. The three trait loci shown are Ag (anther color), PcP (polycarpel), and Fc (flower color). There are multiple Prunus sequences that are anchored to the same position of the TxE Prunus map. The two Populus or Arabidopsis genes are linked by straight lines when the two genes are matched to the same Prunus sequences and by dotted lines when the two genes are matched to different Prunus sequences that are anchored to the same position. The approximate length in peach genome is calculated from the TxE map size 524 cM [39] and the peach genome size 290 Mb [43]. teny to the corresponding parts of LG_II that is reported to be homologus to either to LG_V or LG_XIV. Interestingly, the block in the BAC contig ctg468 was syntenic to regions in Populus LG_II and LG_V that are each 1.7 Mb distant from the regions that were syntenic to peach BAC pPn31C7 ( Figure 5B, Fig 7B). This suggests that BAC contig ctg468 and the peach BAC pPn31C7 may be linked. We did not detect any Prunus blocks sharing more than Prunus blocks that match to more than one site in the Populus genome Figure 7 Prunus blocks that match to more than one site in the Populus genome. The two conserved syntenic groups are selected when more than three Prunus sequences had matches with two different regions in Populus or Arabidopsis. (A) A region in G5 of the Prunus TxE map matches to regions in LG_II and LG_XIV. (B) A region in peach BAC contig ctg468 matches to regions in LG_II and LG_V. Also shown is a block in peach BAC pPn31C7 that is syntenic to regions in LG_II and LG_V, each region is 1.7 Mb apart from the corresponding region that is syntenic to ctg468. The approximate base pair length in peach genome of the syntenic region in genetic and physical map is calculated from the TxE map size 524 cM [39] and the peach genome size 290 Mb [43], and the total physical length of the peach physical map, 303 Mb, [29] and the total unit length of the BAC contigs, 242772.
three sequences and matching to two different regions even though we observed many cases of multiple Arabidopsis regions matching to similar Prunus regions. All the multiple Arabidopsis regions that match to similar regions in Prunus share none or less than three sequences, suggesting Arabidopsis genome underwent severe gene loss after multiple genome duplication.

Syntenic regions around agronomically important trait loci in the Prunus TxE map
The position of 28 major trait loci affecting agronomic characters found in various Prunus species have been previously established in the TxE map using the data from different linkage maps anchored with the TxE reference map [3]. We were interested in finding any syntenic regions conserved between the TxE Prunus map and the Arabidopsis or Populus genomes covering these trait loci. Several syntenic regions matched to trait-loci containing regions in G3 of the Prunus TxE map ( Figure 6). The trait loci contained in these syntenic regions are Ag for anther color of peach and almond (yellow/anthocyanic), PcP for the polycarpel trait of peach [34], and Fc for flower color (pink/pale pink) in peach [35]. All these syntenic regions span 8.3 cM to 15 cM in the TxE map and contained either two or three trait loci ( Figure 6). Among the four Populus blocks that matched to these regions, two blocks in LG_XII and LG_XIV seem to represent duplicated blocks, since two genes matched to the same peach sequences and two genes matched to the sequences that are anchored to the same position in the TxE map. The other two Populus blocks in LG_I and LG_XII may also represent duplicated regions since one of the genes matched to the same peach sequence and the rest matched to the sequences that are anchored to the same position in the TxE map. There were seven Arabidopsis blocks that matched to these regions. Some blocks share one to two genes that match to the same peach sequences and other blocks share two to three genes that match to the sequences that are anchored to the same position in the TxE map. The complex network of synteny in Arabidopsis in this region is also indicative of multiple independent genome duplications and rearrangements in the Arabidopsis genome.

Simulation study
To determine whether the syntenic groups we report were detected by chance, we tested the statistical significance for each group. Both Populus and Arabidopsis genomes were randomized by leaving the locations the same but permuting the gene names. We analyzed 1000 simulated Populus and Arabidopsis genomes for the occurrence of each conserved syntenic group and calculated the probability of the match occurring by chance. The probability of the association by chance was less than 0.1% for all the syntenic groups between Arabidopsis or Populus and the Prunus physical map, less than 1% for groups between Populus and the Prunus genetic map, and less than 5% for the groups between Arabidopsis and the Prunus genetic map. The numbers of syntenic groups at various significance thresholds are shown in Table 4.

Discussion
We compared the level of conserved synteny between the Prunus genome and the whole genome sequences of two model organisms, Arabidopsis and Populus, and the partially sequenced Medicago genome. Since the whole genome sequence of peach or other Prunus sequences is not currently available, we employed available Prunus BAC sequences to assess the level of conserved microsynteny and map-anchored Prunus sequences to assess the pattern of synteny throughout the genome. In the Populus genome, we found well-conserved syntenic regions in all the BACs studied, and the syntenic regions cover almost the entire length of the BACs in some cases. All the BACs showed conserved synteny with two regions in two different Populus chromosomes, supporting the proposed event of the whole genome duplication in Populus [26]. The conserved gene content in the two different Populus genomes showed that different sets of genes were lost in the duplicated Populus regions. The order of genes in these BACs and both of the Populus regions were very well conserved, without signs of genome translocations. The synteny quality within each block was also considerable. On the contrary, we did not detect that level of microsynteny between Prunus BACs and the Arabidopsis genome. The two microsyntenic regions between these contained only four and five gene matches. We also found well-conserved syntenic regions between Prunus BACs and the partially  10 10 10 10 peach-physical/Arabidopsis (pA) 17 17 17 17 sequenced Medicago genome. Interestingly, the homologous BACs of plum, peach, and apricot detected three syntenic regions in three different chromosomes of Medicago. These three regions may be indicative of large-scale or segmental duplication events in the evolutionary history of Medicago, in addition to the whole genome duplication event proposed to have occurred within the rosid I clade.
The other two peach BACs detected only one syntenic region in Medicago. This may be explained by the fact that the Medicago Genome Release 1.0 constitutes only 38-47% of the entire genome. Another possibility is that these peach BACs do have only one conserved syntenic region in Medicago genome. It has shown that the level of internal synteny within both Medicago and Lotus is much lower than the intergenomic synteny between the two, presumably through significant gene loss and rearrangement after the whole genome duplication event [11].
Our study analyzed a fine-level microsynteny between Prunus and the three model dicot organisms. Our results substantiate our previous findings that the level of geneorder conservation between peach and Arabidopsis is very fragmentary [17,36]. The microsynteny between the Prunus and Populus genome, however, is much better conserved, promising the utility of the Populus genome in the study of the Prunus genome and vice versa when the peach genome is sequenced (underway by the Joint Genome Institute). Our results also suggest that the Prunus genome may be closer to Populus than to Medicago, which is evolutionarily closer to Prunus than Populus. Similar results have been reported; two melon BACs showed more conserved synteny to Populus than to the evolutionarily closer Medicago [27]. Lai et al. [28] reported that ordered papaya BAC end sequences showed a higher level of synteny with poplar than with the more closely related Arabidopsis which both belong to the Brassicales.
We also employed Prunus sequences that are anchored to the Prunus genetic map or the peach physical map to study the level of conserved synteny at the whole genome level.
Only the completely sequenced genomes of Populus and Arabidopsis were used in this analysis. A number of syntenic regions that contain several gene pairs were detected between Prunus map anchored sequences and both Populus and Arabidopsis genomes. The number of the syntenic regions was actually higher in Arabidopsis than in Populus. However, in most cases, multiple Arabidopsis regions were syntenic to the overlapping regions in the Prunus TxE map or peach BAC contigs, resulting in a complex network of synteny. In contrast, the syntenic regions in Populus detected more distinct regions in Prunus. We found blocks in both the Prunus genetic map and peach physical map that were syntenic to two different homologous regions in Populus genome. However, we did not find any Prunus regions that detected homologous regions in the Arabidop-sis genome despite the fact that multiple Arabidopsis genomic regions were syntenic to overlapping Prunus regions. Our findings are consistent with previous reports that the Arabidopsis genome went through multiple large genome duplication events followed by frequent gene loss [37,38]. The multiple Arabidopsis genome regions with synteny to overlapping Prunus regions, which we observed, may represent the duplicated regions that subsequently went through gene loss. Simillion et al. [37] have reported that high frequency of gene loss after duplication in Arabidopsis reduces collinearity and that, at extreme, the duplicated regions no longer share homologous genes. They have shown that they could detect these highly degenerated duplicated blocks by indirect comparison with other segments. The synteny obscured by severe subsequent gene loss can impede the transfer of genomic knowledge from one species to another, suggesting the potential problem of using Arabidopsis in the study of other species such as those in Prunus. In contrast, the Populus genome is reported to be a more stable and homologous genome; blocks that have arisen from the most recent genome-wide duplication event can be clearly detected [26]. Our results corroborate these previous findings and suggest that Populus is better model to study synteny with Prunus genomes.

Conclusion
We report the evidence of well-conserved microsynteny between Prunus BACs and two plant model species, Populus and Medicago. The observed network of synteny supported the whole genome duplication events, proposed to have occurred in Populus and Medicago. Interestingly, the level of synteny conservation seemed higher between Prunus-Populus than in Prunus-Medicago, even though Medicago, rather than Populus, is evolutionarily closer to Prunus. In comparison, the level of conserved synteny between the Prunus BAC and Arabidopsis was insignificant. We also analyzed the extent of conserved synteny between Prunus map-anchored sequences and the completely sequenced genomes of Populus and Arabidopsis. We detected a number of syntenic regions that contain several gene pairs between Prunus map anchored sequences and both Populus and Arabidopsis genomes. In comparison to the synteny between Populus and Prunus, we observed a more complex network of synteny between Arabidopsis and Prunus in which multiple Arabidopsis regions shows synteny to overlapping regions in Prunus.

Data Acquisition and Annotation
Three sets of Prunus sequences were used in the analysis of conserved synteny between Prunus and the whole genome sequences of Arabidopsis and Populus: Prunus BAC sequences, Prunus sequences anchored to the Prunus reference map [1,39], and the peach EST sequences anchored to the peach physical map [29]. Prunus BAC sequences were also compared with the partially sequenced Medicago genome [11].
Prunus sequences that are anchored to the TxE Prunus reference map were downloaded from GDR. The majority of the 1093 anchored sequences were derived from peach fruit ESTs [32]. Some ESTs were directly anchored to the map by co-hybridization to BACs with genetic markers, and others were indirectly anchored through hybridization to BACs that belong to the same BAC contig that contain anchored genetic markers. The positions (cM) of the genetic markers were used as the positions for the genetically anchored ESTs on the map. The set also included sequences of markers that are directly used in mapping.
The third set, also downloaded from GDR, comprised of 2140 EST sequences that were anchored to 1500 BAC contigs of the peach physical map. The position of the individual BACs in the BAC contigs were used as the positions ESTs in the peach physical map. For the ESTs that are anchored to multiple overlapping BACs in a BAC contig, the innermost left and right positions were assigned.
The sequence data (TAIR6_pep_20060907) and the chromosome coordinate data (sv_gene.data) of the Arabidopsis translated proteins were downloaded from the Arabidopsis Information Resources (TAIR) database [41]. The sequence data (Poptr1_FilteredModels_proteins.fa) and the chromosome coordinate data(Poptr1_FilteredModels.gff) of the Populus translated proteins were downloaded from the JGI web site http://genome.jgi-psf.org/Poptr1/Poptr1.down load.ftp.html. Medicago genome release version 1.0 data was downloaded from http://medicago.org. International Medicago Genome Annotation Group (IMGAG) annotated protein sequences (MTpep1.txt) were used for our analysis.

Detection of conserved syntenic regions
The predicted protein sequences of the Prunus BACs were compared with the Arabidopsis, Populus, and Medicago proteins by pairwise comparison using the BLASTP program.
The top ten matches with an E value less than 10 -6 were used for further analysis. Syntenic groups were selected when the distance between the two adjacent matches were less than 200 kb and when the syntenic regions contain at least four gene pairs.
Mapped Prunus sequences that are homologous to the Arabidopsis or Populus proteins were determined by pairwise comparison using the BLASTX program. The top ten matches with E value less than 10 -6 were used for further analysis. The syntenic groups were selected when the distance between the two adjacent matches were less than the maximum distance and when the syntenic regions contain at least four gene pairs. For the synteny analysis between the TxE genetic map and Populus or Arabidopsis, the maximum distance was set as the approximate genomic distance that covers about 100 genes -1 Mb for Populus and 0.5 Mb for Arabidopsis. This was approximated by the genome size, 550 Mb for Populus [26] and 157 Mb for Arabidopsis [42], and the predicted number of genes, 45,555 for Populus (annotation v1.1) and 32,041 (TAIR7 Genome release) for Arabidopsis. For sequences anchored in the TxE Prunus genetic map, we selected a less strict parameter, 5 cM, as the maximum distance since most of the sequences are not directly mapped, but are indirectly anchored to the position by the association with other mapped markers (see above). 5 cM is 0.95% of the TxE map (524 cM) [39] and 0.5 Mb that we used for Arabidopsis is about 0.35% of the genome (157 Mb). We used a smaller maximum distance -0.5 Mb for Populus and 0.25 Mb for Arabidopsis -for the synteny analysis between the peach physical map and Populus or Arabidopsis, since the sequences are positioned to BAC contigs, which are much smaller than chromosomes or linkage groups. The maximum distance in peach BAC contig was set to 200 unit lengths (unit given in the FPC output), which corresponds to 0.25 Mb. One unit length corresponds approximately to 1.25 kb, estimated from the total physical length of the peach physical map, 303 Mb, [29] and the total unit length of the BAC contigs, 242772.
We employed DAGchainer [31] to detect collinear segments conserved in Prunus and other genomes of Arabidopsis, Medicago and Populus. When we used mapanchored Prunus sequences, we found many cases in which multiple collinear segments, detected by the software, reside in the same or overlapping genome regions. This occurs when the two genomes in comparison contain syntenic regions in which gene contents are conserved but the gene order is not well conserved, which is caused by differential small genome rearrangements such as conversions, translocations and duplications in the evolutionary history of two genomes. In these regions, DAGchainer can detect more than one collinear syntenic region in the same genomic block by selecting the gene pairs with the conserved order. To solve this problem, we merged the overlapping syntenic regions that are generated by DAGchainer. The syntenic groups were merged when they overlap or when they are separated by less than the maximum distance between the two adjacent matches in both genomes. The merged syntenic groups represent syntenic regions in which the gene contents are conserved but not the gene order. This procedure provides us with a more accurate assessment of the degree of conserved synteny between the two genomes. The Prunus blocks that match to more than one site in the Populus or Arabidopsis genome were detected by selecting syntenic regions that share more than three Prunus sequences. All the in-house developed scripts are available to the public when requested.

Evaluation of the conserved syntenic regions
To determine whether the syntenic groups we report were detected by chance, we tested the statistical significance for each group. Both of the Populus and Arabidopsis genomes were randomized by leaving the locations the same but permuting the gene names. We analyzed 1000 simulated Populus and Arabidopsis genomes for the occurrence of each conserved syntenic group and calculated the probability of the match occurring by chance.

Authors' contributions
SJ designed the protocol for synteny analysis and the statistical analysis, designed and developed scripts, performed the research, analyzed the data and wrote the paper. DJ originally sequenced four Prunus BACs that contain a putative disease resistant gene and participated in the design of the study. IC and TL wrote the scripts for parsing the DAGchainer outputs to further restrict the gap size between the genes in a syntenic region, to merge overlapping syntenic regions, to name the syntenic regions, and to report the results. AA conceived of the study and critically revised the manuscript. BS participated in the design of the study and critically revised the manuscript. DM conceived of the study and participated in its design and coordination, and critically revised the manuscript.