The Medicago Genome Provides Insight into the Evolution of Rhizobial Symbioses

Abstract

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation ¹. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Mya). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species ². Medicago truncatula (Mt) is a long-established model for the study of legume biology. Here we describe the draft sequence of the Mt euchromatin based on a recently completed BAC-assembly supplemented with Illumina-shotgun sequence, together capturing ~94% of all Mt genes. A whole-genome duplication (WGD) approximately 58 Mya played a major role in shaping the Mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the Mt genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max (Gm) and Lotus japonicus (Lj). Mt is a close relative of alfalfa (M. sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the Mt genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox.

Based on optical mapping, the eight pseudomolecules of assembly Mt3.5 span a physical distance of 375 million base pairs (Mb), while fluorescence in situ hybridization indicates they extend from pericentromeres almost to telomeric ends (Figures S1, S2). Altogether, Mt3.5 consists of 2,536 BACs (Tables S1, S2) with 273 physical gaps (including centromeres) (Table S3) and 101 internal sequencing gaps. The pseudomolecules contain 246 Mb of nonredundant sequence (Table S2) located entirely within the optical map (Figure S3). Another 146 unfinished BACs/BAC pools that cannot be placed on the optical map contribute 17.3 Mb. Regions not represented in pseudomolecules or unanchored BACs were captured through assembly of ~40x coverage Illumina sequencing, yielding 104.2 Mb of additional unique sequence. Though not directly tested, the Illumina sequence is expected to lie predominantly within the boundaries of pseudomolecules (See below). Based on EST alignments, the combined datasets capture ~94% of expressed genes, providing a highly informative, though still draft stage platform for analyzing the euchromatin of Mt.

Altogether there are 62,388 gene loci in Mt3.5 (Table S4, Figure S4) with 14,322 gene predictions annotated as transposons. Pseudomolecules and unassigned BACs contain a total of 44,124 gene loci, 177,271 retroelement-related regions, and 26,487 DNA transposons, while non-redundant Illumina assemblies contribute an additional 18,264 genes, 75,777 retrotransposon regions, and 8,476 DNA transposons (Tables S5-S9) along with 1,418 organellar insertions (Datafile S1). For pseudomolecules and unassigned BACs, this translates to 16.8 genes, 67.6 retrotransposons and 10.1 DNA transposons per 100 kbp. Within Illumina sequence assemblies, gene density (17.1 per 100 kbp) and retrotransposon density (72.2 per 100 kbp) are similar to pseudomolecules and unassigned BACs, while DNA transposon density is somewhat lower (8.2 per 100 kbp). Similarities in gene and transposon densities between BAC and Illumina sequences support the assertion that the Illumina sequence is euchromatic, though the possibility that some Illumina assemblies come from low copy regions within heterochromatin can not be excluded. Considering only the 47,845 genes with experimental or database support (Table S4), the average Mt gene is 2,211 bp in length, contains 4.0 exons, and has a CDS of 1,001 bp. These values are similar to those observed previously in Arabidopsis thaliana (At) (2,174 bp), Oryza sativa (3,403 bp) and Populus trichocarpa (Pt) (2,301 bp) ^4-6.

Recent analyses of plant genomes indicate a shared whole genome hexaploidy (WGH) preceding the rosid-asterid split at 140-150 Mya ⁷. Duplication patterns and genomic comparisons strongly suggest an additional WGD ~58 Mya in the papilionoids ^{8, 9}. Near the time of this WGD, papilionoids radiated into several clades, the largest of which split quickly into two subclades, the Hologalegina (including Mt and Lj) and the milletioids (including Gm and other phaseoloids) at ~54 Mya ². We therefore compared Mt pseudomolecules with other sequenced plant genomes to learn more about shared synteny and genome duplication history.

There is significant macrosynteny among Mt, Lj and Gm (Figure 1, Figure S5a-b). Conserved blocks, sometimes as large as chromosome arms, span most euchromatin in all three genomes. A given Mt region is typically syntenic with one other Mt region as a result of the ~58 Mya WGD, usually in small blocks showing degraded synteny (Figure 2, Figure S6). A given Mt region is most similar to two Gm regions via speciation at ~54 Mya and the Glycine WGD at <13 Mya ¹⁰ and less similar to two other Gm regions resulting from the ~58 Mya and <13 Mya WGD events. A Mt region is likewise most similar to one Lj region via speciation at ~50 Mya and somewhat less similar to a second Lj region as a result of the ~58 Mya WGD. Finally, each Mt region and its homoeologue typically exhibit similarity to three Vitis vinifera (Vv) regions via the pre-Rosid WGH. Exceptions to these patterns could be due to gene losses, gains, or rearrangements specific to the Mt lineage, resulting in synteny being more evident between Mt and other genomes than in self-comparisons. Indeed, self-comparisons within Mt reveal few remnants of the legume specific WGD (Figure 2, Figures S6). While this seems paradoxical, it is probably explained by extensive gene fractionation between WGD-derived homoeologues in Mt. In Figure 3, two short regions on Mt01 and Mt03 resulting from the ~58 Mya WGD are displayed beside microsyntenic regions of Gm and Vv. As expected, many genes are microsyntenic between Mt and Gm (ranging from 7/19 between Mt03 and Gm14 to 10/20 between Mt01 and Gm17). Between the two Mt homoeologues, however, only 6 out of 33 genes (or collapsed gene families) are microsyntenic, with a homoeologue missing from one or the other duplicate (Table S10). Apparently, there have been many more changes, large and small, in Mt than in Gm since the legume WGD. This is borne out by the fact that synteny blocks in Mt are one-third the length of those remaining from the papilionoid WGD in Gm (524 kb vs. 1503 kb) with the average number of homologous gene pairs per block correspondingly lower (12.4 vs. 31.0).

Figure 1. Circos diagram illustrating syntentic relationships between Medicago, Glycine, Lotus and Vitis.

Homologous gene pairs were identified for all pairwise comparisons between Mt, Gm, Lj and Vv genomes. Syntenic regions associated with the ancestral WGD events were identified by visually inspection of correponding dot-plots. The large Mt5–Mt8 synteny block (yellow) was found to have two syntenic regions in Lj (red), four syntenic regions in Gm (blue) and three in Vv (green).

Figure 2. Circos diagram illustrating the Medicago WGD and selected gene families.

The 963 WGD-derived paralogous gene pairs were examined for overlap with the nodule-enhanced gene list (Datafile S2). Resulting gene pairs were joined and plotted as either blue circles (only one of the duplicates is nodule-enhanced) or red (both nodule enhanced). Gene densities of NBS-LRRs, NCRs and other defensin-like proteins are plotted against chromosome position. Density was calculated using a sliding window (100 kb window with 50 kb steps).

Figure 3. Microsynteny comparison between Medicago homoeologues and corresponding regions of Glycine and Vitis.

Microsyntenic genome segments are centered around Medtr3g104510/Medtr1g015890 (Table S10), a duplicated region derived from the ~58 Mya WGD event noted in orange. The <13 Mya Gm-specific WGD is colored yellow. Orthologous/paralogous gene pairs are indicated through use of a common color. White arrows represent genes with no syntenic homologue(s) in this genome region. Gray arrow indicates a Mt gene model with syntenic homologue in soybean, but no soybean gene model in the current annotation (www.phytozome.net/soybean).

The Mt genome also has undergone high rates of local gene duplication. The ratio of related genes within local clusters compared to all genes in families is 0.339 in Mt, 3.1-fold higher than in Gm and 1.6-fold higher than in At or Pt. (“Local clusters” are defined as genes in a family all within 100 gene models of one another.) The excess of local gene duplications in Mt is observed genome-wide and affects many families. There are 2.63 times as many gene families with local duplications in Mt compared with Gm (2,980 vs. 1,131), an excess that also is seen in detailed comparisons of syntenic regions in Mt and Gm. We examined 16.3 Mbp of Mt05 showing synteny to two large regions of Gm01 plus homoeologous blocks on Gm02, Gm09 and Gm11. In these regions, 25.8% of Mt genes are locally duplicated compared with just 8.0% in Gm. Local gene duplications and losses have contributed both to synteny disruptions (Figure 3, Figure S7) and to high gene count (62,388) in Mt — a value nearly as high as the 65,781 total gene models in Gm despite its additional (<13 Mya) WGD. Local gene duplications are evident in certain gene families, such as F-box genes, which have undergone dramatic expansions (Figure S8, Table S11). Mt also has experienced higher rates of base substitution compared to other plant genomes (Figure S9). Assuming 58 Mya as the date of the legume WGD, then the rate of synonymous substitutions per site per year (ss/s/yr) in Mt is 1.08 × 10⁻⁸, 1.8 times faster than estimates in other vascular plants ¹¹. Higher rates of mutation and greater levels of rearrangement in Mt following the papilionoid duplication may have been driven by factors including short generation times, high selfing rates or small effective population sizes, though these characteristics are not unique to Mt.

Legumes and actinorhizal species are capable of forming a specialized organ, the root nodule, a highly differentiated structure hosting nitrogen-fixing symbionts. Phylogenetic studies suggest that nodulation may have evolved multiple times in the Fabidae, but the observation that all nodulating species are contained within this single clade implies a predisposition to nodulate evolved in their common ancestor ¹². It is unknown whether nodulation with rhizobia preceded the divergence of the three legume subfamilies or evolved on multiple occassions ¹³. Nevertheless, rhizobial nodulation and the 58 Mya WGD are features common to most papilionoid legumes and both occurred early in the emergence of the group ². Given that WGDs generate genetic redundancy that potentially facilitates the emergence of novel gene functions without compromising existing ones ¹⁴, we examined the Mt genome to ask whether the 58 Mya WGD might have played a role in the evolution of rhizobial nodulation in Mt and its relatives.

Nod factors are bacterial signalling molecules that initiate nodulation. Previous studies have shown that several of the plant components involved in the response to Nod factors also function in mycorrhizal signaling ¹⁵. However, some Nod factor receptors and transcription factors (TFs) have distinctly nodulation-specific functions. Among these nodulation-specific components, we found the Nod factor receptor, NFP, and the transcription factor, ERN1, each have paralogs, LYR1 and ERN2 respectively, that trace back to the papilionoid WGD based on genome location and Ks values (Figure S10, Datafile S2). Both sets of gene pairs also exhibit contrasting expression patterns and functional specialization. NFP and ERN1 are expressed predominantly in the nodule and are known to function in nodulation ^{16, 17}, while LYR1 and ERN2 are highly expressed during mycorrhizal colonization (Figure S11). These observations indicate that two important nodulation-specific signaling components in Mt might have evolved from more ancient genes originally functioning in mycorrhizal signaling and then duplicated by the 58 Mya WGD. In the case of MtNFP/MtLYR1, this conclusion is supported by the observation that the apparent ortholog of NFP in the nodulating non-legume Parasponia andersonii functions in both nodule and mycorrhizal signaling ¹⁸. Thus, the 58 Mya WGD appears to have led to sub-functionalization of an ancestral gene participating in both interactions, resulting in two homoeologous genes that each performs just one of the original functions.

To further assess the contribution of the WGD to Mt nodulation, we analyzed expression of paralogous gene pairs using RNA-seq data from six organs (Supplemental Methods S5.1). A total of 963 WGD-derived gene pairs were found (Datafile S2) with 618 pairs (1,046 genes) having RNA-seq data for one or both homoeologue. We then determined the number of genes showing organ-enhanced expression (defined as genes with expression level in a single organ at least twice the level in any other) within the pseudomolecule and the WGD-derived gene sets (Table S12). In both cases, different organs contained markedly different numbers of genes with enhanced expression (Χ² with 5 df, p = 1E-272), however the rank order among the organs was identical. Roots exhibited the largest number of genes with enhanced expression followed by flower, nodule, leaf, seed/pod and bud. Among gene pairs with nodule-enhanced expression, both paralogs were nodule-enhanced in eight pairs, while just a single paralog was nodule-enhanced in the other 43 pairs. This is consistent with nodulation predating the WGD and further sub- and neo-functionalization emerging afterwards. We went on to examine TFs since they can act as regulators of plant growth and development. A total of 3,692 putative TF genes were discovered (Datafile S3), representing 5.9% of all Mt gene models (Table S13). Of the 1,513 TF genes on pseudomolecules with RNA-seq data, 142 genes (9.4%) derived from the 58 Mya WGD (Figure S12, Datafile S4), consistent with previous observations indicating greater retention of TFs following polyploidy ¹⁹. Nodule-enhanced expression was significantly higher among TFs (92/1,513 or 6.1%) than among all pseudomolecule genes (1,111/23,478 or 4.7%) (Χ² with 1 df, p = 0.024) (Table S12). Nodule-enhanced expression was even higher in WGD-derived TFs (11/142 or 7.7%), although this enrichment did not reach statistical significance (p = 0.113). As expected, ERN1 is found within this group of WGD-retained, nodule-enhanced TFs.

These results show that many paralogous genes retained from the 58 Mya WGD, especially signaling components and regulators, have undergone sub- or neo-functionalization, including several with specialized roles in nodulation. Nevertheless, separate phylogenetic analyses (Supplemental Methods S5.5) indicate that some nodule-related genes derive from the more ancient pre-Rosid WGH, with their nodule-related functions pre-dating the 58 Mya WGD (Datafile S5). Taken together, these results are consistent with a model where the capacity for primitive interaction with new symbionts derived from existing mycorrhizal machinery involving genes recruited from the pre-Rosid WGH. This capacity would have arisen early in the Fabidae clade and led to the appearance of nodulation in multiple lineages ^{13, 20}. Later, the 58 Mya WGD would have resulted in additional genes, including NFP, ERN1 and the TFs described above, that went on to become specialized for nodule-related functions in the Papilionoideae.

Medicago contains additional amplified gene families, many nodulation-related and found in tandem clusters. Mt has nine symbiotic leghemoglobins, more than twice the number in Lj or Gm (Figure S13). Five of these genes are located in a tight cluster on Mt5. The Mt genome contains 593 nodule cysteine rich peptides (NCRs) (Datafile S6), a gene family restricted to Mt and its relatives ²¹. NCRs are noteworthy because they include members essential for terminal differentiation of rhizobia ²². NCRs are tightly clustered within the Mt genome (Figure 2), with 75% found in clusters of up to 11 members. The Mt genome also has 764 NBS-LRR genes (Datafile S7), more than other sequenced plant genomes to date ^23-25, many with nodule-specific expression (Figure S14). Almost 90% of NBS-LRRs occur in clusters and genome regions showing limited macrosynteny to other species, such as Mt3 and Mt6, are locations of large NBS-LRR superclusters (Figure 2, Tables S14, S15). Finally, Mt secretes flavonoid signaling molecules to induce the nod genes of Sinorhizobium meliloti ²⁶. In Mt, the corresponding biosynthetic pathway has expanded dramatically, with 28 Mt chalcone synthase genes in clusters of up to seven members compared to just four chalcone synthases in At ²⁷ (Datafile S8). Mt has ten chalcone reductases compared to none in At ²⁸ and Mt has 11 chalcone isomerase genes, including one cluster of seven members, compared to just one representative in At ²⁹ (Figures S15, S16).

In summary, analysis of the Mt genome supports earlier studies indicating that the dramatic radiation of the legume family (at least the papilionoid subfamily) is partly attributed to the 58 Mya WGD ³⁰. Our results suggest that the WGD early in papilionoid evolution allowed the emergence of critical components in Nod factor signaling and contributed to the complexity of rhizobial nodulation observed in this clade. As such, the WGD appears to have played a crucial role in the success of papilionoid legumes, enhancing their utility to humans.

METHODS SUMMARY

DNA sequencing

Six A17 BAC and one fosmid library were used to create Mt3.5 (Table S1). Most were processed by Sanger paired-end sequencing of 3-6 kb shotgun libraries. Sequences were downloaded in February/March 2009 with scaffolding performed by aligning all BAC and fosmid ends against contigs and then anchored and ordered primarily by optical mapping. Separately, 25 Gb of Illumina sequence was generated using short (375 nt) inserts plus 2.1 Gb from a 5 kb mate-pair library, then assembled using CLCbio (www.clcbio.com) and Soap (http://soap.genomics.org.cn/).

RNA sequencing

Five tissues were used for RNA-seq analysis with ~10 million Illumina 36 bp reads per library (Table S12). Three tissues were used for small RNA analysis with ~3 million reads per Illumina library (Figures S17-S18, Table S16, Datafile S9).